Modified polynucleotides for the production of oncology-related proteins and peptides

ABSTRACT

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of oncology-related polynucleotides, oncology-related primary transcripts and oncology-related mmRNA molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/106,988, filed Dec. 16, 2013, entitled Modified PolynucleotidesEncoding Apoptosis Inducing Factor 1, which is a continuation ofInternational Patent Application No PCT/US2013/030070, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production ofOncology-Related Proteins and Peptides which claims priority to U.S.Provisional Patent Application No. 61/681,742, filed, Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Oncology-RelatedProteins and Peptides, U.S. Provisional Patent Application No.61/737,224, filed Dec. 14, 2012, entitled Terminally Optimized ModifiedRNAs, International Application No PCT/US2012/069610, filed Dec. 14,2012, entitled Modified Nucleoside, Nucleotide, and Nucleic AcidCompositions, U.S. Provisional Patent Application No. 61/618,862, filedApr. 2, 2012, entitled Modified Polynucleotides for the Production ofBiologics, U.S. Provisional Patent Application No. 61/681,645, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofBiologics, U.S. Provisional Patent Application No. 61/737,130, filedDec. 14, 2012, entitled Modified Polynucleotides for the Production ofBiologics, U.S. Provisional Patent Application No. 61/618,866, filedApr. 2, 2012, entitled Modified Polynucleotides for the Production ofAntibodies, U.S. Provisional Patent Application No. 61/681,647, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofAntibodies, U.S. Provisional Patent Application No. 61/737,134, filedDec. 14, 2012, entitled Modified Polynucleotides for the Production ofAntibodies, U.S. Provisional Patent Application No. 61/618,868, filedApr. 2, 2012, entitled Modified Polynucleotides for the Production ofVaccines, U.S. Provisional Patent Application No. 61/681,648, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofVaccines, U.S. Provisional Patent Application No. 61/737,135, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofVaccines, U.S. Provisional Patent Application No. 61/618,870, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofTherapeutic Proteins and Peptides, U.S. Provisional Patent ApplicationNo. 61/681,649, filed Aug. 10, 2012, entitled Modified Polynucleotidesfor the Production of Therapeutic Proteins and Peptides, U.S.Provisional Patent Application No. 61/737,139, filed Dec. 14, 2012,Modified Polynucleotides for the Production of Therapeutic Proteins andPeptides, U.S. Provisional Patent Application No. 61/618,873, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins, U.S. Provisional Patent Application No. 61/681,650,filed Aug. 10, 2012, entitled Modified Polynucleotides for theProduction of Secreted Proteins, U.S. Provisional Patent Application No.61/737,147, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Secreted Proteins, U.S. Provisional Patent ApplicationNo. 61/618,878, filed Apr. 2, 2012, entitled Modified Polynucleotidesfor the Production of Plasma Membrane Proteins, U.S. Provisional PatentApplication No. 61/681,654, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Plasma Membrane Proteins, U.S.Provisional Patent Application No. 61/737,152, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins, U.S. Provisional Patent Application No. 61/618,885, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/681,658, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins, U.S. Provisional Patent Application No. 61/737,155, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/618,896, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins, U.S. Provisional Patent Application No. 61/668,157, filed Jul.5, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/681,661, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins, U.S. Provisional Patent Application No. 61/737,160, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/618,911, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Nuclear Proteins, U.S. ProvisionalPatent Application No. 61/681,667, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Nuclear Proteins, U.S.Provisional Patent Application No. 61/737,168, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins, U.S. Provisional Patent Application No. 61/618,922, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/681,675, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/737,174, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/618,935, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/681,687, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/737,184, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/618,945, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/681,696, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/737,191, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/618,953, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/681,704, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/737,203, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/618,961, filed Apr. 2, 2012, entitled Dosing Methodsfor Modified mRNA, U.S. Provisional Patent Application No. 61/648,286,filed May 17, 2012, entitled Dosing Methods for Modified mRNA, thecontents of each of which are herein incorporated by reference in itsentirety.

This application is also related to International Publication No.PCT/US2012/58519, filed Oct. 3, 2012, entitled Modified Nucleosides,Nucleotides, and Nucleic Acids, and Uses Thereof and InternationalPublication No. PCT/US2012/69610, filed Dec. 14, 2012, entitled ModifiedNucleoside, Nucleotide, and Nucleic Acid Compositions.

The instant application is also related to co-pending applications, eachfiled concurrently herewith on Mar. 9, 2013 and having Attorney DocketNumber M300.20, (PCT/US13/030062) entitled Modified Polynucleotides forthe Production of Biologics and Proteins Associated with Human Disease;Attorney Docket Number M301.20, (PCT/US13/030063) entitled ModifiedPolynucleotides; Attorney Docket Number M304.20 (PCT/US13/030064),entitled Modified Polynucleotides for the Production of SecretedProteins; Attorney Docket Number M304.20 (PCT/US13/030064), entitledModified Polynucleotides for the Production of Secreted Proteins;Attorney Docket Number M305.20 (PCT/US13/030059), entitled ModifiedPolynucleotides for the Production of Membrane Proteins; Attorney DocketNumber M306.20 (PCT/US13/030066), entitled Modified Polynucleotides forthe Production of Cytoplasmic and Cytoskeletal Proteins; Attorney DocketNumber M308.20 (PCT/US13/030067), entitled Modified Polynucleotides forthe Production of Nuclear Proteins; Attorney Docket Number M309.20(PCT/US13/030060), entitled Modified Polynucleotides for the Productionof Proteins; Attorney Docket Number M310.20 (PCT/US13/030061), entitledModified Polynucleotides for the Production of Proteins Associated withHuman Disease; and Attorney Docket Number MNC1.20 (PCT/US13/030068),entitled Modified Polynucleotides for the Production of CosmeticProteins and Peptides, the contents of each of which are hereinincorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing file entitledMNC2USCON6SQLST.txt, created on Feb. 4, 2016 which is 91,364,237 Bytesin size. The information in electronic format of the sequence listing isincorporated by reference in its entirety.

REFERENCE TO LENGTHY TABLE

The specification includes a lengthy Table 6. Lengthy Table 6 has beensubmitted via EFS-Web in electronic format as follows: File name:MNC2TBL.txt, Date

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160152678A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).created: Feb. 4, 2016; File size: 538,729 Bytes and is incorporatedherein by reference in its entirety. Please refer to the end of thespecification for access instructions.

FIELD OF THE INVENTION

The invention relates to compositions, methods, processes, kits anddevices for the design, preparation, manufacture and/or formulation ofoncology-related polynucleotides, primary constructs and modified mRNAmolecules (mmRNA).

BACKGROUND OF THE INVENTION

Cancer is a disease characterized by uncontrolled cell division andgrowth within the body. In the United States, roughly a third of allwomen and half of all men will experience cancer in their lifetime.Polypeptides are involved in every aspect of the disease includingcancer cell biology (carcinogenesis, cell cycle suppression, DNA repairand angiogenesis), treatment (immunotherapy, hormone manipulation,enzymatic inhibition), diagnosis and determination of cancer type(molecular markers for breast, prostate, colon and cervical cancer forexample). With the host of undesired consequences brought about bystandard treatments such as chemotherapy and radiotherapy used today,genetic therapy for the manipulation of disease-related peptidesprovides a more targeted approach to disease diagnosis, treatment andmanagement.

To this end, the inventors have shown that certain modified mRNAsequences have the potential as therapeutics with benefits beyond justevading, avoiding or diminishing the immune response. Such studies aredetailed in published co-pending applications International ApplicationPCT/US2011/046861 filed Aug. 5, 2011 and PCT/US2011/054636 filed Oct. 3,2011, International Application number PCT/US2011/054617 filed Oct. 3,2011, the contents of which are incorporated herein by reference intheir entirety.

The present invention addresses this need by providing nucleic acidbased compounds or polynucleotide-encoding nucleic acid-based compounds(e.g., modified mRNA or mmRNA) which encode an oncology-relatedpolypeptide of interest and which have structural and/or chemicalfeatures that avoid one or more of the problems in the art.

SUMMARY OF THE INVENTION

Described herein are compositions, methods, processes, kits and devicesfor the design, preparation, manufacture and/or formulation of modifiedmRNA (mmRNA) molecules encoding at least one oncology-relatedpolypeptide of interest.

The present invention provides a method of treating a disease, disorderand/or condition in a subject in need thereof by increasing the level ofan oncology-related polypeptide of interest comprising administering tosaid subject an isolated polynucleotide encoding said oncology-relatedpolypeptide. The disease, disorder and/or condition may include, but isnot limited to, adrenal cortical cancer, advanced cancer, anal cancer,aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bonemetastasis, brain tumors, brain cancer, breast cancer, childhood cancer,cancer of unknown primary origin, Castleman disease, cervical cancer,colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing familyof tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoidtumors, gastrointestinal stromal tumors, gestational trophoblasticdisease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma,laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, chronic myeloidleukemia, chronic myelomonocytic leukemia, liver cancer, non-small celllung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma ofthe skin, malignant mesothelioma, multiple myeloma, myelodysplasticsyndrome, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity andoropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,penile cancer, pituitary tumors, prostate cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue,basal and squamous cell skin cancer, melanoma, small intestine cancer,stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroidcancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrommacroglobulinemia, Wilms tumor and secondary cancers caused by cancertreatment.

The present invention provides a method of reducing, eliminating, orpreventing tumor growth in a subject in need thereof by increasing thelevel of an oncology-related polypeptide of interest comprisingadministering to said subject an isolated polynucleotide encoding saidoncology-related polypeptide. The tumor growth may be associated with orresults from a disease, disorder and/or condition such as, but notlimited to, adrenal cortical cancer, advanced cancer, anal cancer,aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bonemetastasis, brain tumors, brain cancer, breast cancer, childhood cancer,cancer of unknown primary origin, Castleman disease, cervical cancer,colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing familyof tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoidtumors, gastrointestinal stromal tumors, gestational trophoblasticdisease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma,laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, chronic myeloidleukemia, chronic myelomonocytic leukemia, liver cancer, non-small celllung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma ofthe skin, malignant mesothelioma, multiple myeloma, myelodysplasticsyndrome, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity andoropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,penile cancer, pituitary tumors, prostate cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue,basal and squamous cell skin cancer, melanoma, small intestine cancer,stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroidcancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrommacroglobulinemia, Wilms tumor and secondary cancers caused by cancertreatment.

The present invention provides a method of reducing and/or amerioratingat least on symptom of cancer in a subject in need thereof by increasingthe level of an oncology-related polypeptide of interest comprisingadministering to said subject an isolated polynucleotide encoding saidoncology-related polypeptide.

The present invention provides an isolated polynucleotide comprising afirst region of linked nucleosides, said first region encoding theoncology-related polypeptide of interest, a first flanking regionlocated at the 5′ terminus of said first region comprising a sequence oflinked nucleosides selected from the group consisting of the native 5′untranslated region (UTR) of any of SEQ ID NOs: 4704-9203, SEQ ID NOs:1-4 and functional variants thereof and a second flanking region locatedat the 3′ terminus of said first region comprising a sequence of linkednucleosides selected from the group consisting of the native 3′ UTR ofany of SEQ ID NOs: 4704-9203, SEQ ID NOs: 5-21 and functional variantsthereof and a 3′ tailing sequence of linked nucleosides. The isolatedpolynucleotide may further be substantially purified.

Further, the first region of the isolated polynucleotides of the presentmay comprise two stop codons. In one embodiment, the first regionfurther comprises a first stop codon “TGA” and a second stop codonselected from the group consisting of “TAA,” “TGA” and “TAG.”

The 3′ tailing sequence of the isolated polynucleotides of the presentinvention may further include linked nucleosides such as, but notlimited to, a poly-A tail of approximately 130 nucleotides and a polyA-G quartet.

The first flanking region of the isolated polynucleotide may furthercomprise at least one 5′ terminal cap such as, but not limited to, Cap0,Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,and 2-azido-guanosine.

The nucleotides of the isolated polynucleotides of the present inventionmay comprise at least two modifications and a translatable region. Themodification may be on at least one nucleoside and/or the backbone ofsaid nucleotides, on both a nucleoside and a backbone linkage or on asugar of the nucleoside. The modification may comprise replacing atleast one phosphodiester linkage with a phosphorothioate linkage. Themodification may include, but are not limited to, pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. Thebackbone linkage may be modified by the replacement of one or moreoxygen atoms. The nucleobase modified may be selected from, but is notlimited to, cytosine, guanine, adenine, thymine and uracil.

The isolated polynucleotides of the present invention may be formulated.The formulation may comprise a lipid which may include, but is notlimited to, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200,DLin-MC3-DMA, PLGA, PEG, PEG-DMG, PEGylated lipids and mixtures thereof.

The isolated polynucleotides of the present invention may beadministered at a total daily dose of between 0.001 ug and 150 ug. Theadministration may be by injection, topical administration, ophthalmicadministration and intranasal administration. The injection may includeinjections such as, but not limited to, intradermal, subcutaneous andintramuscular. The topical administration may be, but is not limited to,a cream, lotion, ointment, gel, spray, solution and the like. Thetopical administration may further include a penetration enhancer suchas, but not limited to, surfactants, fatty acids, bile salts, chelatingagents, non-chelating non-surfactants, polyoxyethylene-9-lauryl ether,polyoxyethylene-20-cetyl ether, fatty acids and/or salts in combinationwith bile acids and/or salts, sodium salt in combination with lauricacid, capric acid and UDCA, and the like.

The isolated polynucleotide formulation may further include apharmaceutically acceptable excipient such as, but not limited to, asolvent, aqueous solvent, non-aqueous solvent, dispersion media,diluent, dispersion, suspension aid, surface active agent, isotonicagent, thickening or emulsifying agent, preservative, lipid, lipidoidsliposome, lipid nanoparticle, core-shell nanoparticles, polymer,lipoplex, peptide, protein, cell, hyaluronidase, and mixtures thereof.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 is a schematic of an oncology related-primary construct of thepresent invention.

FIG. 2 illustrates lipid structures in the prior art useful in thepresent invention. Shown are the structures for 98N12-5 (TETA5-LAP),DLin-DMA, DLin-K-DMA(2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA,DLin-MC3-DMA and C12-200.

FIG. 3 is a representative plasmid useful in the IVT reactions taughtherein. The plasmid contains Insert 64818, designed by the instantinventors.

FIG. 4 is a gel profile of modified mRNA encapsulated in PLGAmicrospheres.

DETAILED DESCRIPTION

It is of great interest in the fields of therapeutics, diagnostics,reagents and for biological assays to be able to deliver a nucleic acid,e.g., a ribonucleic acid (RNA) inside a cell, whether in vitro, in vivo,in situ or ex vivo, such as to cause intracellular translation of thenucleic acid and production of an encoded polypeptide of interest. Ofparticular importance is the delivery and function of a non-integrativepolynucleotide.

Described herein are compositions (including pharmaceuticalcompositions) and methods for the design, preparation, manufactureand/or formulation of polynucleotides encoding one or moreoncology-related polypeptides of interest. Also provided are systems,processes, devices and kits for the selection, design and/or utilizationof the polynucleotides encoding the oncology-related polypeptides ofinterest described herein.

According to the present invention, these oncology-relatedpolynucleotides are preferably modified as to avoid the deficiencies ofother polypeptide-encoding molecules of the art. Hence thesepolynucleotides are referred to as modified mRNA or mmRNA.

The use of modified polynucleotides in the fields of antibodies,viruses, veterinary applications and a variety of in vivo settings hasbeen explored by the inventors and these studies are disclosed in forexample, co-pending and co-owned U.S. provisional patent applicationSer. Nos. 61/470,451 filed Mar. 31, 2011 teaching in vivo applicationsof mmRNA; 61/517,784 filed on Apr. 26, 2011 teaching engineered nucleicacids for the production of antibody polypeptides; 61/519,158 filed May17, 2011 teaching veterinary applications of mmRNA technology;61/533,537 filed on Sep. 12, 2011 teaching antimicrobial applications ofmmRNA technology; 61/533,554 filed on Sep. 12, 2011 teaching viralapplications of mmRNA technology, 61/542,533 filed on Oct. 3, 2011teaching various chemical modifications for use in mmRNA technology;61/570,690 filed on Dec. 14, 2011 teaching mobile devices for use inmaking or using mmRNA technology; 61/570,708 filed on Dec. 14, 2011teaching the use of mmRNA in acute care situations; 61/576,651 filed onDec. 16, 2011 teaching terminal modification architecture for mmRNA;61/576,705 filed on Dec. 16, 2011 teaching delivery methods usinglipidoids for mmRNA; 61/578,271 filed on Dec. 21, 2011 teaching methodsto increase the viability of organs or tissues using mmRNA; 61/581,322filed on Dec. 29, 2011 teaching mmRNA encoding cell penetratingpeptides; 61/581,352 filed on Dec. 29, 2011 teaching the incorporationof cytotoxic nucleosides in mmRNA and 61/631,729 filed on Jan. 10, 2012teaching methods of using mmRNA for crossing the blood brain barrier;all of which are herein incorporated by reference in their entirety.

Provided herein, in part, are oncology-related polynucleotides,oncology-related primary constructs and/or oncology-related mmRNAencoding oncology-related polypeptides of interest which have beendesigned to improve one or more of the stability and/or clearance intissues, receptor uptake and/or kinetics, cellular access by thecompositions, engagement with translational machinery, mRNA half-life,translation efficiency, immune evasion, protein production capacity,secretion efficiency (when applicable), accessibility to circulation,protein half-life and/or modulation of a cell's status, function and/oractivity.

I. COMPOSITIONS OF THE INVENTION (MMRNA)

The present invention provides nucleic acid molecules, specificallyoncology-related polynucleotides, oncology-related primary constructsand/or oncology-related mmRNA which encode one or more oncology-relatedpolypeptides of interest. The term “nucleic acid,” in its broadestsense, includes any compound and/or substance that comprise a polymer ofnucleotides. These polymers are often referred to as polynucleotides.Exemplary nucleic acids or polynucleotides of the invention include, butare not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNAhaving a β-D-ribo configuration, α-LNA having an α-L-ribo configuration(a diastereomer of LNA), 2′-amino-LNA having a 2′-aminofunctionalization, and 2′-amino-α-LNA having a 2′-aminofunctionalization) or hybrids thereof.

In preferred embodiments, the nucleic acid molecule is a messenger RNA(mRNA). As used herein, the term “messenger RNA” (mRNA) refers to anyoncology-related polynucleotide which encodes an oncology-relatedpolypeptide of interest and which is capable of being translated toproduce the encoded oncology-related polypeptide of interest in vitro,in vivo, in situ or ex vivo.

Traditionally, the basic components of an mRNA molecule include at leasta coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. Buildingon this wild type modular structure, the present invention expands thescope of functionality of traditional mRNA molecules by providingoncology-related polynucleotides or oncology-related primary RNAconstructs which maintain a modular organization, but which comprise oneor more structural and/or chemical modifications or alterations whichimpart useful properties to the polynucleotide including, in someembodiments, the lack of a substantial induction of the innate immuneresponse of a cell into which the oncology-related polynucleotide isintroduced. As such, modified mRNA molecules of the present inventionare termed “mmRNA.” As used herein, a “structural” feature ormodification is one in which two or more linked nucleotides areinserted, deleted, duplicated, inverted or randomized in anoncology-related polynucleotide, oncology-related primary construct oroncology-related mmRNA without significant chemical modification to thenucleotides themselves. Because chemical bonds will necessarily bebroken and reformed to effect a structural modification, structuralmodifications are of a chemical nature and hence are chemicalmodifications. However, structural modifications will result in adifferent sequence of nucleotides. For example, the polynucleotide“ATCG” may be chemically modified to “AT-5meC-G”. The samepolynucleotide may be structurally modified from “ATCG” to “ATCCCG”.Here, the dinucleotide “CC” has been inserted, resulting in a structuralmodification to the oncology-related polynucleotide.

mmRNA Architecture

The mmRNA of the present invention are distinguished from wild type mRNAin their functional and/or structural design features which serve to, asevidenced herein, overcome existing problems of effective polypeptideproduction using nucleic acid-based therapeutics.

FIG. 1 shows a representative polynucleotide primary construct 100 ofthe present invention. As used herein, the term “primary construct” or“primary mRNA construct” refers to an oncology-related polynucleotidetranscript which encodes one or more oncology-related polypeptides ofinterest and which retains sufficient structural and/or chemicalfeatures to allow the oncology-related polypeptide of interest encodedtherein to be translated. Oncology-related primary constructs may beoncology-related polynucleotides of the invention. When structurally orchemically modified, the oncology-related primary construct may bereferred to as an oncology-related mmRNA.

Returning to FIG. 1, the oncology-related primary construct 100 herecontains a first region of linked nucleotides 102 that is flanked by afirst flanking region 104 and a second flaking region 106. As usedherein, the “first region” may be referred to as a “coding region” or“region encoding” or simply the “first region.” This first region mayinclude, but is not limited to, the encoded oncology-related polypeptideof interest. The oncology-related polypeptide of interest may compriseat its 5′ terminus one or more signal sequences encoded by a signalsequence region 103. The flanking region 104 may comprise a region oflinked nucleotides comprising one or more complete or incomplete 5′ UTRssequences. The flanking region 104 may also comprise a 5′ terminal cap108. The second flanking region 106 may comprise a region of linkednucleotides comprising one or more complete or incomplete 3′ UTRs. Theflanking region 106 may also comprise a 3′ tailing sequence 110.

Bridging the 5′ terminus of the first region 102 and the first flankingregion 104 is a first operational region 105. Traditionally thisoperational region comprises a start codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a start codon.

Bridging the 3′ terminus of the first region 102 and the second flankingregion 106 is a second operational region 107. Traditionally thisoperational region comprises a stop codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a stop codon. According to the present invention, multipleserial stop codons may also be used. In one embodiment, the operationregion of the present invention may comprise two stop codons. The firststop codon may be “TGA” and the second stop codon may be selected fromthe group consisting of “TAA,” “TGA” and “TAG.”

Generally, the shortest length of the first region of theoncology-related primary construct of the present invention can be thelength of a nucleic acid sequence that is sufficient to encode for adipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide,a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. Inanother embodiment, the length may be sufficient to encode a peptide of2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 aminoacids. The length may be sufficient to encode for a peptide of at least11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that isno longer than 40 amino acids, e.g. no longer than 35, 30, 25, 20, 17,15, 14, 13, 12, 11 or 10 amino acids. Examples of dipeptides that thepolynucleotide sequences can encode or include, but are not limited to,carnosine and anserine.

Generally, the length of the first region encoding the oncology-relatedpolypeptide of interest of the present invention is greater than about30 nucleotides in length (e.g., at least or greater than about 35, 40,45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400,1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000,50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000nucleotides). As used herein, the “first region” may be referred to as a“coding region” or “region encoding” or simply the “first region.”

In some embodiments, the oncology-related polynucleotide,oncology-related primary construct, or oncology-related mmRNA includesfrom about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000,from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000,from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000,from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000,from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to100,000).

According to the present invention, the first and second flankingregions may range independently from 15-1,000 nucleotides in length(e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,and 1,000 nucleotides).

According to the present invention, the tailing sequence may range fromabsent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Wherethe tailing region is a polyA tail, the length may be determined inunits of or as a function of polyA binding protein binding. In thisembodiment, the polyA tail is long enough to bind at least 4 monomers ofpolyA binding protein. PolyA binding protein monomers bind to stretchesof approximately 38 nucleotides. As such, it has been observed thatpolyA tails of about 80 nucleotides and 160 nucleotides are functional.

According to the present invention, the capping region may comprise asingle cap or a series of nucleotides forming the cap. In thisembodiment the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7,1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In someembodiments, the cap is absent.

According to the present invention, the first and second operationalregions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or30 or fewer nucleotides in length and may comprise, in addition to astart and/or stop codon, one or more signal and/or restrictionsequences.

Cyclic mmRNA

According to the present invention, an oncology-related primaryconstruct or oncology-related mmRNA may be cyclized, or concatemerized,to generate a translation competent molecule to assist interactionsbetween poly-A binding proteins and 5′-end binding proteins. Themechanism of cyclization or concatemerization may occur through at least3 different routes: 1) chemical, 2) enzymatic, and 3) ribozymecatalyzed. The newly formed 5′-/3′-linkage may be intramolecular orintermolecular.

In the first route, the 5′-end and the 3′-end of the nucleic acid maycontain chemically reactive groups that, when close together, form a newcovalent linkage between the 5′-end and the 3′-end of the molecule. The5′-end may contain an NETS-ester reactive group and the 3′-end maycontain a 3′-amino-terminated nucleotide such that in an organic solventthe 3′-amino-terminated nucleotide on the 3′-end of a synthetic mRNAmolecule will undergo a nucleophilic attack on the 5′-NHS-ester moietyforming a new 5′-/3′-amide bond.

In the second route, T4 RNA ligase may be used to enzymatically link a5′-phosphorylated nucleic acid molecule to the 3′-hydroxyl group of anucleic acid forming a new phosphorodiester linkage. In an examplereaction, 1 μg of a nucleic acid molecule is incubated at 37° C. for 1hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich,Mass.) according to the manufacturer's protocol. The ligation reactionmay occur in the presence of a split oligonucleotide capable ofbase-pairing with both the 5′- and 3′-region in juxtaposition to assistthe enzymatic ligation reaction.

In the third route, either the 5′- or 3′-end of the cDNA templateencodes a ligase ribozyme sequence such that during in vitrotranscription, the resultant nucleic acid molecule can contain an activeribozyme sequence capable of ligating the 5′-end of a nucleic acidmolecule to the 3′-end of a nucleic acid molecule. The ligase ribozymemay be derived from the Group I Intron, Group I Intron, Hepatitis DeltaVirus, Hairpin ribozyme or may be selected by SELEX (systematicevolution of ligands by exponential enrichment). The ribozyme ligasereaction may take 1 to 24 hours at temperatures between 0 and 37° C.

mmRNA Multimers

According to the present invention, multiple distinct oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA may be linked together through the 3′-end using nucleotides whichare modified at the 3′-terminus. Chemical conjugation may be used tocontrol the stoichiometry of delivery into cells. For example, theglyoxylate cycle enzymes, isocitrate lyase and malate synthase, may besupplied into HepG2 cells at a 1:1 ratio to alter cellular fatty acidmetabolism. This ratio may be controlled by chemically linkingoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA using a 3′-azido terminated nucleotide on oneoncology-related polynucleotide, oncology-related primary construct oroncology-related mmRNA species and a C5-ethynyl or alkynyl-containingnucleotide on the opposite oncology-related polynucleotide,oncology-related primary construct or oncology-related mmRNA species.The modified nucleotide is added post-transcriptionally using terminaltransferase (New England Biolabs, Ipswich, Mass.) according to themanufacturer's protocol. After the addition of the 3′-modifiednucleotide, the two oncology-related polynucleotide, oncology-relatedprimary construct or oncology-related mmRNA species may be combined inan aqueous solution, in the presence or absence of copper, to form a newcovalent linkage via a click chemistry mechanism as described in theliterature.

In another example, more than two oncology-related polynucleotides maybe linked together using a functionalized linker molecule. For example,a functionalized saccharide molecule may be chemically modified tocontain multiple chemical reactive groups (SH—, NH₂—, N₃, etc. . . . )to react with the cognate moiety on a 3′-functionalized oncology-relatedmRNA molecule (i.e., a 3′-maleimide ester, 3′-NHS-ester, alkynyl). Thenumber of reactive groups on the modified saccharide can be controlledin a stoichiometric fashion to directly control the stoichiometric ratioof conjugated oncology-related polynucleotide, oncology-related primaryconstruct or oncology-related mmRNA.

mmRNA Conjugates and Combinations

In order to further enhance oncology-related protein production,oncology-related primary constructs or oncology-related mmRNA of thepresent invention can be designed to be conjugated to otherpolynucleotides, oncology-related polypeptides, dyes, intercalatingagents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases, proteins, e.g., glycoproteins, orpeptides, e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell, hormones and hormonereceptors, non-peptidic species, such as lipids, lectins, carbohydrates,vitamins, cofactors, or a drug.

Conjugation may result in increased stability and/or half life and maybe particularly useful in targeting the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA to specific sites in the cell, tissue or organism.

According to the present invention, the oncology-related mmRNA oroncology-related primary constructs may be administered with, or furtherencode one or more of RNAi agents, siRNAs, shRNAs, miRNAs, miRNA bindingsites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that inducetriple helix formation, aptamers or vectors, and the like.

Bifunctional mmRNA

In one embodiment of the invention are bifunctional oncology-relatedpolynucleotides (e.g., bifunctional oncology-related primary constructsor oncology-related bifunctional mmRNA). As the name implies,bifunctional oncology-related polynucleotides are those having orcapable of at least two functions. These molecules may also byconvention be referred to as multi-functional.

The multiple functionalities of bifunctional oncology-relatedpolynucleotides may be encoded by the RNA (the function may not manifestuntil the encoded product is translated) or may be a property of thepolynucleotide itself. It may be structural or chemical. Bifunctionalmodified oncology-related polynucleotides may comprise a function thatis covalently or electrostatically associated with the polynucleotides.Further, the two functions may be provided in the context of a complexof a mmRNA and another molecule.

Bifunctional oncology-related polynucleotides may encodeoncology-related peptides which are anti-proliferative. These peptidesmay be linear, cyclic, constrained or random coil. They may function asaptamers, signaling molecules, ligands or mimics or mimetics thereof.Anti-proliferative peptides may, as translated, be from 3 to 50 aminoacids in length. They may be 5-40, 10-30, or approximately 15 aminoacids long. They may be single chain, multichain or branched and mayform complexes, aggregates or any multi-unit structure once translated.

Noncoding Oncology-Related Polynucleotides and Primary Constructs

As described herein, provided are oncology-related polynucleotides andoncology-related primary constructs having sequences that are partiallyor substantially not translatable, e.g., having a noncoding region. Suchnoncoding region may be the “first region” of the oncology-relatedprimary construct. Alternatively, the noncoding region may be a regionother than the first region. Such molecules are generally nottranslated, but can exert an effect on protein production by one or moreof binding to and sequestering one or more translational machinerycomponents such as a ribosomal protein or a transfer RNA (tRNA), therebyeffectively reducing protein expression in the cell or modulating one ormore pathways or cascades in a cell which in turn alters protein levels.The oncology-related polynucleotide and/or oncology-related primaryconstruct may contain or encode one or more long noncoding RNA (lncRNA,or lincRNA) or portion thereof, a small nucleolar RNA (sno-RNA), microRNA (miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA(piRNA).

Oncology-Related Polypeptides of Interest

According to the present invention, the oncology-related primaryconstruct is designed to encode one or more oncology-relatedpolypeptides of interest or fragments thereof. An oncology-relatedpolypeptide of interest may include, but is not limited to, wholepolypeptides, a plurality of polypeptides or fragments of polypeptides,which independently may be encoded by one or more nucleic acids, aplurality of nucleic acids, fragments of nucleic acids or variants ofany of the aforementioned. As used herein, the term “oncology-relatedpolypeptides of interest” refers to any polypeptide which is selected tobe encoded in the oncology-related primary construct of the presentinvention. As used herein, “polypeptide” means a polymer of amino acidresidues (natural or unnatural) linked together most often by peptidebonds. The term, as used herein, refers to proteins, polypeptides, andpeptides of any size, structure, or function. In some instances thepolypeptide encoded is smaller than about 50 amino acids and thepolypeptide is then termed a peptide. If the polypeptide is a peptide,it will be at least about 2, 3, 4, or at least 5 amino acid residueslong. Thus, polypeptides include gene products, naturally occurringpolypeptides, synthetic polypeptides, homologs, orthologs, paralogs,fragments and other equivalents, variants, and analogs of the foregoing.A polypeptide may be a single molecule or may be a multi-molecularcomplex such as a dimer, trimer or tetramer. They may also comprisesingle chain or multichain polypeptides such as antibodies or insulinand may be associated or linked. Most commonly disulfide linkages arefound in multichain polypeptides. The term polypeptide may also apply toamino acid polymers in which one or more amino acid residues are anartificial chemical analogue of a corresponding naturally occurringamino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and preferably, they will be at least about 80%, morepreferably at least about 90% identical (homologous) to a native orreference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains one or more amino acids whichwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, e.g., phenylalanine may act as an inactivatingsubstitution for tyrosine; or alanine may act as an inactivatingsubstitution for serine.

“Homology” as it applies to amino acid sequences is defined as thepercentage of residues in the candidate amino acid sequence that areidentical with the residues in the amino acid sequence of a secondsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well known in the art. It is understoodthat homology depends on a calculation of percent identity but maydiffer in value due to gaps and penalties introduced in the calculation.

By “homologs” as it applies to polypeptide sequences means thecorresponding sequence of other species having substantial identity to asecond sequence of a second species.

“Analogs” is meant to include polypeptide variants which differ by oneor more amino acid alterations, e.g., substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present invention contemplates several types of compositions whichare polypeptide based including variants and derivatives. These includesubstitutional, insertional, deletion and covalent variants andderivatives. The term “derivative” is used synonymously with the term“variant” but generally refers to a molecule that has been modifiedand/or changed in any way relative to a reference molecule or startingmolecule.

As such, mmRNA encoding oncology-related polypeptides containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences, in particular theoncology-related polypeptide sequences disclosed herein, are includedwithin the scope of this invention. For example, sequence tags or aminoacids, such as one or more lysines, can be added to the peptidesequences of the invention (e.g., at the N-terminal or C-terminal ends).Sequence tags can be used for peptide purification or localization.Lysines can be used to increase peptide solubility or to allow forbiotinylation. Alternatively, amino acid residues located at the carboxyand amino terminal regions of the amino acid sequence of a peptide orprotein may optionally be deleted providing for truncated sequences.Certain amino acids (e.g., C-terminal or N-terminal residues) mayalternatively be deleted depending on the use of the sequence, as forexample, expression of the sequence as part of a larger sequence whichis soluble, or linked to a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions may be single, where only one amino acid inthe molecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Insertional variants” when referring to polypeptides are those with oneor more amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved. Ordinarily, deletional variants will have one or more aminoacids deleted in a particular region of the molecule.

“Covalent derivatives” when referring to polypeptides includemodifications of a native or starting protein with an organicproteinaceous or non-proteinaceous derivatizing agent, and/orpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theprotein with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-proteinantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed oncology-related polypeptide.Glutaminyl and asparaginyl residues are frequently post-translationallydeamidated to the corresponding glutamyl and aspartyl residues.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues may be present in theoncology-related polypeptides produced in accordance with the presentinvention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

“Features” when referring to polypeptides are defined as distinct aminoacid sequence-based components of a molecule. Features of thepolypeptides encoded by the mmRNA of the present invention includesurface manifestations, local conformational shape, folds, loops,half-loops, domains, half-domains, sites, termini or any combinationthereof.

As used herein when referring to polypeptides the term “surfacemanifestation” refers to a polypeptide based component of a proteinappearing on an outermost surface.

As used herein when referring to polypeptides the term “localconformational shape” means a polypeptide based structural manifestationof a protein which is located within a definable space of the protein.

As used herein when referring to polypeptides the term “fold” refers tothe resultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of energetic forces.Regions formed in this way include hydrophobic and hydrophilic pockets,and the like.

As used herein the term “turn” as it relates to protein conformationmeans a bend which alters the direction of the backbone of a peptide orpolypeptide and may involve one, two, three or more amino acid residues.

As used herein when referring to polypeptides the term “loop” refers toa structural feature of a polypeptide which may serve to reverse thedirection of the backbone of a peptide or polypeptide. Where the loop isfound in a polypeptide and only alters the direction of the backbone, itmay comprise four or more amino acid residues. Oliva et al. haveidentified at least 5 classes of protein loops (J. Mol Biol 266 (4):814-830; 1997). Loops may be open or closed. Closed loops or “cyclic”loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsbetween the bridging moieties. Such bridging moieties may comprise acysteine-cysteine bridge (Cys-Cys) typical in polypeptides havingdisulfide bridges or alternatively bridging moieties may be non-proteinbased such as the dibromozylyl agents used herein.

As used herein when referring to polypeptides the term “half-loop”refers to a portion of an identified loop having at least half thenumber of amino acid resides as the loop from which it is derived. It isunderstood that loops may not always contain an even number of aminoacid residues. Therefore, in those cases where a loop contains or isidentified to comprise an odd number of amino acids, a half-loop of theodd-numbered loop will comprise the whole number portion or next wholenumber portion of the loop (number of amino acids of the loop/2+/−0.5amino acids). For example, a loop identified as a 7 amino acid loopcould produce half-loops of 3 amino acids or 4 amino acids(7/2=3.5+/−0.5 being 3 or 4).

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the term “half-domain”means a portion of an identified domain having at least half the numberof amino acid resides as the domain from which it is derived. It isunderstood that domains may not always contain an even number of aminoacid residues. Therefore, in those cases where a domain contains or isidentified to comprise an odd number of amino acids, a half-domain ofthe odd-numbered domain will comprise the whole number portion or nextwhole number portion of the domain (number of amino acids of thedomain/2+/−0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/−0.5 being 3 or 4). It is also understood thatsubdomains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids that compriseany of the domain types herein need not be contiguous along the backboneof the polypeptide (i.e., nonadjacent amino acids may fold structurallyto produce a domain, half-domain or subdomain).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a peptide or polypeptide that may be modified,manipulated, altered, derivatized or varied within the polypeptide basedmolecules of the present invention.

As used herein the terms “termini” or “terminus” when referring topolypeptides refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide to be encoded by the oncology-related primaryconstruct or oncology-related mmRNA of the invention, any of severalmanipulations and/or modifications of these features may be performed bymoving, swapping, inverting, deleting, randomizing or duplicating.Furthermore, it is understood that manipulation of features may resultin the same outcome as a modification to the molecules of the invention.For example, a manipulation which involved deleting a domain wouldresult in the alteration of the length of a molecule just asmodification of a nucleic acid to encode less than a full lengthmolecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis. Theresulting modified molecules may then be tested for activity using invitro or in vivo assays such as those described herein or any othersuitable screening assay known in the art.

According to the present invention, the oncology-related polypeptidesmay comprise a consensus sequence which is discovered through rounds ofexperimentation. As used herein a “consensus” sequence is a singlesequence which represents a collective population of sequences allowingfor variability at one or more sites.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of oncology-related polypeptides of interest of thisinvention. For example, provided herein is any protein fragment (meaningan oncology-related polypeptide sequence at least one amino acid residueshorter than a reference oncology-related polypeptide sequence butotherwise identical) of a reference oncology-related protein 10, 20, 30,40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length.In another example, any oncology-related protein that includes a stretchof about 20, about 30, about 40, about 50, or about 100 amino acidswhich are about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, or about 100% identical to any of the sequencesdescribed herein can be utilized in accordance with the invention. Incertain embodiments, a polypeptide to be utilized in accordance with theinvention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided or referenced herein.

Encoded Oncology-Related Polypeptides

The oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be designed to encode oncology-relatedpolypeptides of interest such as oncology-related peptides and proteins.

In one embodiment oncology-related primary constructs oroncology-related mmRNA of the present invention may encode variantpolypeptides which have a certain identity with a referenceoncology-related polypeptide sequence. As used herein, a “referenceoncology-related polypeptide sequence” refers to a startingoncology-related polypeptide sequence. Reference sequences may be wildtype sequences or any sequence to which reference is made in the designof another sequence. A “reference polypeptide sequence” may, e.g., beany one of SEQ ID NOs: 4704-9203 as disclosed herein, e.g., any of SEQID NOs 4704, 4705, 4706, 4707, 4708, 4709, 4710, 4711, 4712, 4713, 4714,4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722, 4723, 4724, 4725, 4726,4727, 4728, 4729, 4730, 4731, 4732, 4733, 4734, 4735, 4736, 4737, 4738,4739, 4740, 4741, 4742, 4743, 4744, 4745, 4746, 4747, 4748, 4749, 4750,4751, 4752, 4753, 4754, 4755, 4756, 4757, 4758, 4759, 4760, 4761, 4762,4763, 4764, 4765, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4773, 4774,4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786,4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798,4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4808, 4809, 4810,4811, 4812, 4813, 4814, 4815, 4816, 4817, 4818, 4819, 4820, 4821, 4822,4823, 4824, 4825, 4826, 4827, 4828, 4829, 4830, 4831, 4832, 4833, 4834,4835, 4836, 4837, 4838, 4839, 4840, 4841, 4842, 4843, 4844, 4845, 4846,4847, 4848, 4849, 4850, 4851, 4852, 4853, 4854, 4855, 4856, 4857, 4858,4859, 4860, 4861, 4862, 4863, 4864, 4865, 4866, 4867, 4868, 4869, 4870,4871, 4872, 4873, 4874, 4875, 4876, 4877, 4878, 4879, 4880, 4881, 4882,4883, 4884, 4885, 4886, 4887, 4888, 4889, 4890, 4891, 4892, 4893, 4894,4895, 4896, 4897, 4898, 4899, 4900, 4901, 4902, 4903, 4904, 4905, 4906,4907, 4908, 4909, 4910, 4911, 4912, 4913, 4914, 4915, 4916, 4917, 4918,4919, 4920, 4921, 4922, 4923, 4924, 4925, 4926, 4927, 4928, 4929, 4930,4931, 4932, 4933, 4934, 4935, 4936, 4937, 4938, 4939, 4940, 4941, 4942,4943, 4944, 4945, 4946, 4947, 4948, 4949, 4950, 4951, 4952, 4953, 4954,4955, 4956, 4957, 4958, 4959, 4960, 4961, 4962, 4963, 4964, 4965, 4966,4967, 4968, 4969, 4970, 4971, 4972, 4973, 4974, 4975, 4976, 4977, 4978,4979, 4980, 4981, 4982, 4983, 4984, 4985, 4986, 4987, 4988, 4989, 4990,4991, 4992, 4993, 4994, 4995, 4996, 4997, 4998, 4999, 5000, 5001, 5002,5003, 5004, 5005, 5006, 5007, 5008, 5009, 5010, 5011, 5012, 5013, 5014,5015, 5016, 5017, 5018, 5019, 5020, 5021, 5022, 5023, 5024, 5025, 5026,5027, 5028, 5029, 5030, 5031, 5032, 5033, 5034, 5035, 5036, 5037, 5038,5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046, 5047, 5048, 5049, 5050,5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058, 5059, 5060, 5061, 5062,5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071, 5072, 5073, 5074,5075, 5076, 5077, 5078, 5079, 5080, 5081, 5082, 5083, 5084, 5085, 5086,5087, 5088, 5089, 5090, 5091, 5092, 5093, 5094, 5095, 5096, 5097, 5098,5099, 5100, 5101, 5102, 5103, 5104, 5105, 5106, 5107, 5108, 5109, 5110,5111, 5112, 5113, 5114, 5115, 5116, 5117, 5118, 5119, 5120, 5121, 5122,5123, 5124, 5125, 5126, 5127, 5128, 5129, 5130, 5131, 5132, 5133, 5134,5135, 5136, 5137, 5138, 5139, 5140, 5141, 5142, 5143, 5144, 5145, 5146,5147, 5148, 5149, 5150, 5151, 5152, 5153, 5154, 5155, 5156, 5157, 5158,5159, 5160, 5161, 5162, 5163, 5164, 5165, 5166, 5167, 5168, 5169, 5170,5171, 5172, 5173, 5174, 5175, 5176, 5177, 5178, 5179, 5180, 5181, 5182,5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191, 5192, 5193, 5194,5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203, 5204, 5205, 5206,5207, 5208, 5209, 5210, 5211, 5212, 5213, 5214, 5215, 5216, 5217, 5218,5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, 5227, 5228, 5229, 5230,5231, 5232, 5233, 5234, 5235, 5236, 5237, 5238, 5239, 5240, 5241, 5242,5243, 5244, 5245, 5246, 5247, 5248, 5249, 5250, 5251, 5252, 5253, 5254,5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264, 5265, 5266,5267, 5268, 5269, 5270, 5271, 5272, 5273, 5274, 5275, 5276, 5277, 5278,5279, 5280, 5281, 5282, 5283, 5284, 5285, 5286, 5287, 5288, 5289, 5290,5291, 5292, 5293, 5294, 5295, 5296, 5297, 5298, 5299, 5300, 5301, 5302,5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311, 5312, 5313, 5314,5315, 5316, 5317, 5318, 5319, 5320, 5321, 5322, 5323, 5324, 5325, 5326,5327, 5328, 5329, 5330, 5331, 5332, 5333, 5334, 5335, 5336, 5337, 5338,5339, 5340, 5341, 5342, 5343, 5344, 5345, 5346, 5347, 5348, 5349, 5350,5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358, 5359, 5360, 5361, 5362,5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370, 5371, 5372, 5373, 5374,5375, 5376, 5377, 5378, 5379, 5380, 5381, 5382, 5383, 5384, 5385, 5386,5387, 5388, 5389, 5390, 5391, 5392, 5393, 5394, 5395, 5396, 5397, 5398,5399, 5400, 5401, 5402, 5403, 5404, 5405, 5406, 5407, 5408, 5409, 5410,5411, 5412, 5413, 5414, 5415, 5416, 5417, 5418, 5419, 5420, 5421, 5422,5423, 5424, 5425, 5426, 5427, 5428, 5429, 5430, 5431, 5432, 5433, 5434,5435, 5436, 5437, 5438, 5439, 5440, 5441, 5442, 5443, 5444, 5445, 5446,5447, 5448, 5449, 5450, 5451, 5452, 5453, 5454, 5455, 5456, 5457, 5458,5459, 5460, 5461, 5462, 5463, 5464, 5465, 5466, 5467, 5468, 5469, 5470,5471, 5472, 5473, 5474, 5475, 5476, 5477, 5478, 5479, 5480, 5481, 5482,5483, 5484, 5485, 5486, 5487, 5488, 5489, 5490, 5491, 5492, 5493, 5494,5495, 5496, 5497, 5498, 5499, 5500, 5501, 5502, 5503, 5504, 5505, 5506,5507, 5508, 5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516, 5517, 5518,5519, 5520, 5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529, 5530,5531, 5532, 5533, 5534, 5535, 5536, 5537, 5538, 5539, 5540, 5541, 5542,5543, 5544, 5545, 5546, 5547, 5548, 5549, 5550, 5551, 5552, 5553, 5554,5555, 5556, 5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565, 5566,5567, 5568, 5569, 5570, 5571, 5572, 5573, 5574, 5575, 5576, 5577, 5578,5579, 5580, 5581, 5582, 5583, 5584, 5585, 5586, 5587, 5588, 5589, 5590,5591, 5592, 5593, 5594, 5595, 5596, 5597, 5598, 5599, 5600, 5601, 5602,5603, 5604, 5605, 5606, 5607, 5608, 5609, 5610, 5611, 5612, 5613, 5614,5615, 5616, 5617, 5618, 5619, 5620, 5621, 5622, 5623, 5624, 5625, 5626,5627, 5628, 5629, 5630, 5631, 5632, 5633, 5634, 5635, 5636, 5637, 5638,5639, 5640, 5641, 5642, 5643, 5644, 5645, 5646, 5647, 5648, 5649, 5650,5651, 5652, 5653, 5654, 5655, 5656, 5657, 5658, 5659, 5660, 5661, 5662,5663, 5664, 5665, 5666, 5667, 5668, 5669, 5670, 5671, 5672, 5673, 5674,5675, 5676, 5677, 5678, 5679, 5680, 5681, 5682, 5683, 5684, 5685, 5686,5687, 5688, 5689, 5690, 5691, 5692, 5693, 5694, 5695, 5696, 5697, 5698,5699, 5700, 5701, 5702, 5703, 5704, 5705, 5706, 5707, 5708, 5709, 5710,5711, 5712, 5713, 5714, 5715, 5716, 5717, 5718, 5719, 5720, 5721, 5722,5723, 5724, 5725, 5726, 5727, 5728, 5729, 5730, 5731, 5732, 5733, 5734,5735, 5736, 5737, 5738, 5739, 5740, 5741, 5742, 5743, 5744, 5745, 5746,5747, 5748, 5749, 5750, 5751, 5752, 5753, 5754, 5755, 5756, 5757, 5758,5759, 5760, 5761, 5762, 5763, 5764, 5765, 5766, 5767, 5768, 5769, 5770,5771, 5772, 5773, 5774, 5775, 5776, 5777, 5778, 5779, 5780, 5781, 5782,5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794,5795, 5796, 5797, 5798, 5799, 5800, 5801, 5802, 5803, 5804, 5805, 5806,5807, 5808, 5809, 5810, 5811, 5812, 5813, 5814, 5815, 5816, 5817, 5818,5819, 5820, 5821, 5822, 5823, 5824, 5825, 5826, 5827, 5828, 5829, 5830,5831, 5832, 5833, 5834, 5835, 5836, 5837, 5838, 5839, 5840, 5841, 5842,5843, 5844, 5845, 5846, 5847, 5848, 5849, 5850, 5851, 5852, 5853, 5854,5855, 5856, 5857, 5858, 5859, 5860, 5861, 5862, 5863, 5864, 5865, 5866,5867, 5868, 5869, 5870, 5871, 5872, 5873, 5874, 5875, 5876, 5877, 5878,5879, 5880, 5881, 5882, 5883, 5884, 5885, 5886, 5887, 5888, 5889, 5890,5891, 5892, 5893, 5894, 5895, 5896, 5897, 5898, 5899, 5900, 5901, 5902,5903, 5904, 5905, 5906, 5907, 5908, 5909, 5910, 5911, 5912, 5913, 5914,5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922, 5923, 5924, 5925, 5926,5927, 5928, 5929, 5930, 5931, 5932, 5933, 5934, 5935, 5936, 5937, 5938,5939, 5940, 5941, 5942, 5943, 5944, 5945, 5946, 5947, 5948, 5949, 5950,5951, 5952, 5953, 5954, 5955, 5956, 5957, 5958, 5959, 5960, 5961, 5962,5963, 5964, 5965, 5966, 5967, 5968, 5969, 5970, 5971, 5972, 5973, 5974,5975, 5976, 5977, 5978, 5979, 5980, 5981, 5982, 5983, 5984, 5985, 5986,5987, 5988, 5989, 5990, 5991, 5992, 5993, 5994, 5995, 5996, 5997, 5998,5999, 6000, 6001, 6002, 6003, 6004, 6005, 6006, 6007, 6008, 6009, 6010,6011, 6012, 6013, 6014, 6015, 6016, 6017, 6018, 6019, 6020, 6021, 6022,6023, 6024, 6025, 6026, 6027, 6028, 6029, 6030, 6031, 6032, 6033, 6034,6035, 6036, 6037, 6038, 6039, 6040, 6041, 6042, 6043, 6044, 6045, 6046,6047, 6048, 6049, 6050, 6051, 6052, 6053, 6054, 6055, 6056, 6057, 6058,6059, 6060, 6061, 6062, 6063, 6064, 6065, 6066, 6067, 6068, 6069, 6070,6071, 6072, 6073, 6074, 6075, 6076, 6077, 6078, 6079, 6080, 6081, 6082,6083, 6084, 6085, 6086, 6087, 6088, 6089, 6090, 6091, 6092, 6093, 6094,6095, 6096, 6097, 6098, 6099, 6100, 6101, 6102, 6103, 6104, 6105, 6106,6107, 6108, 6109, 6110, 6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118,6119, 6120, 6121, 6122, 6123, 6124, 6125, 6126, 6127, 6128, 6129, 6130,6131, 6132, 6133, 6134, 6135, 6136, 6137, 6138, 6139, 6140, 6141, 6142,6143, 6144, 6145, 6146, 6147, 6148, 6149, 6150, 6151, 6152, 6153, 6154,6155, 6156, 6157, 6158, 6159, 6160, 6161, 6162, 6163, 6164, 6165, 6166,6167, 6168, 6169, 6170, 6171, 6172, 6173, 6174, 6175, 6176, 6177, 6178,6179, 6180, 6181, 6182, 6183, 6184, 6185, 6186, 6187, 6188, 6189, 6190,6191, 6192, 6193, 6194, 6195, 6196, 6197, 6198, 6199, 6200, 6201, 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6370,6371, 6372, 6373, 6374, 6375, 6376, 6377, 6378, 6379, 6380, 6381, 6382,6383, 6384, 6385, 6386, 6387, 6388, 6389, 6390, 6391, 6392, 6393, 6394,6395, 6396, 6397, 6398, 6399, 6400, 6401, 6402, 6403, 6404, 6405, 6406,6407, 6408, 6409, 6410, 6411, 6412, 6413, 6414, 6415, 6416, 6417, 6418,6419, 6420, 6421, 6422, 6423, 6424, 6425, 6426, 6427, 6428, 6429, 6430,6431, 6432, 6433, 6434, 6435, 6436, 6437, 6438, 6439, 6440, 6441, 6442,6443, 6444, 6445, 6446, 6447, 6448, 6449, 6450, 6451, 6452, 6453, 6454,6455, 6456, 6457, 6458, 6459, 6460, 6461, 6462, 6463, 6464, 6465, 6466,6467, 6468, 6469, 6470, 6471, 6472, 6473, 6474, 6475, 6476, 6477, 6478,6479, 6480, 6481, 6482, 6483, 6484, 6485, 6486, 6487, 6488, 6489, 6490,6491, 6492, 6493, 6494, 6495, 6496, 6497, 6498, 6499, 6500, 6501, 6502,6503, 6504, 6505, 6506, 6507, 6508, 6509, 6510, 6511, 6512, 6513, 6514,6515, 6516, 6517, 6518, 6519, 6520, 6521, 6522, 6523, 6524, 6525, 6526,6527, 6528, 6529, 6530, 6531, 6532, 6533, 6534, 6535, 6536, 6537, 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6706,6707, 6708, 6709, 6710, 6711, 6712, 6713, 6714, 6715, 6716, 6717, 6718,6719, 6720, 6721, 6722, 6723, 6724, 6725, 6726, 6727, 6728, 6729, 6730,6731, 6732, 6733, 6734, 6735, 6736, 6737, 6738, 6739, 6740, 6741, 6742,6743, 6744, 6745, 6746, 6747, 6748, 6749, 6750, 6751, 6752, 6753, 6754,6755, 6756, 6757, 6758, 6759, 6760, 6761, 6762, 6763, 6764, 6765, 6766,6767, 6768, 6769, 6770, 6771, 6772, 6773, 6774, 6775, 6776, 6777, 6778,6779, 6780, 6781, 6782, 6783, 6784, 6785, 6786, 6787, 6788, 6789, 6790,6791, 6792, 6793, 6794, 6795, 6796, 6797, 6798, 6799, 6800, 6801, 6802,6803, 6804, 6805, 6806, 6807, 6808, 6809, 6810, 6811, 6812, 6813, 6814,6815, 6816, 6817, 6818, 6819, 6820, 6821, 6822, 6823, 6824, 6825, 6826,6827, 6828, 6829, 6830, 6831, 6832, 6833, 6834, 6835, 6836, 6837, 6838,6839, 6840, 6841, 6842, 6843, 6844, 6845, 6846, 6847, 6848, 6849, 6850,6851, 6852, 6853, 6854, 6855, 6856, 6857, 6858, 6859, 6860, 6861, 6862,6863, 6864, 6865, 6866, 6867, 6868, 6869, 6870, 6871, 6872, 6873, 6874,6875, 6876, 6877, 6878, 6879, 6880, 6881, 6882, 6883, 6884, 6885, 6886,6887, 6888, 6889, 6890, 6891, 6892, 6893, 6894, 6895, 6896, 6897, 6898,6899, 6900, 6901, 6902, 6903, 6904, 6905, 6906, 6907, 6908, 6909, 6910,6911, 6912, 6913, 6914, 6915, 6916, 6917, 6918, 6919, 6920, 6921, 6922,6923, 6924, 6925, 6926, 6927, 6928, 6929, 6930, 6931, 6932, 6933, 6934,6935, 6936, 6937, 6938, 6939, 6940, 6941, 6942, 6943, 6944, 6945, 6946,6947, 6948, 6949, 6950, 6951, 6952, 6953, 6954, 6955, 6956, 6957, 6958,6959, 6960, 6961, 6962, 6963, 6964, 6965, 6966, 6967, 6968, 6969, 6970,6971, 6972, 6973, 6974, 6975, 6976, 6977, 6978, 6979, 6980, 6981, 6982,6983, 6984, 6985, 6986, 6987, 6988, 6989, 6990, 6991, 6992, 6993, 6994,6995, 6996, 6997, 6998, 6999, 7000, 7001, 7002, 7003, 7004, 7005, 7006,7007, 7008, 7009, 7010, 7011, 7012, 7013, 7014, 7015, 7016, 7017, 7018,7019, 7020, 7021, 7022, 7023, 7024, 7025, 7026, 7027, 7028, 7029, 7030,7031, 7032, 7033, 7034, 7035, 7036, 7037, 7038, 7039, 7040, 7041, 7042,7043, 7044, 7045, 7046, 7047, 7048, 7049, 7050, 7051, 7052, 7053, 7054,7055, 7056, 7057, 7058, 7059, 7060, 7061, 7062, 7063, 7064, 7065, 7066,7067, 7068, 7069, 7070, 7071, 7072, 7073, 7074, 7075, 7076, 7077, 7078,7079, 7080, 7081, 7082, 7083, 7084, 7085, 7086, 7087, 7088, 7089, 7090,7091, 7092, 7093, 7094, 7095, 7096, 7097, 7098, 7099, 7100, 7101, 7102,7103, 7104, 7105, 7106, 7107, 7108, 7109, 7110, 7111, 7112, 7113, 7114,7115, 7116, 7117, 7118, 7119, 7120, 7121, 7122, 7123, 7124, 7125, 7126,7127, 7128, 7129, 7130, 7131, 7132, 7133, 7134, 7135, 7136, 7137, 7138,7139, 7140, 7141, 7142, 7143, 7144, 7145, 7146, 7147, 7148, 7149, 7150,7151, 7152, 7153, 7154, 7155, 7156, 7157, 7158, 7159, 7160, 7161, 7162,7163, 7164, 7165, 7166, 7167, 7168, 7169, 7170, 7171, 7172, 7173, 7174,7175, 7176, 7177, 7178, 7179, 7180, 7181, 7182, 7183, 7184, 7185, 7186,7187, 7188, 7189, 7190, 7191, 7192, 7193, 7194, 7195, 7196, 7197, 7198,7199, 7200, 7201, 7202, 7203, 7204, 7205, 7206, 7207, 7208, 7209, 7210,7211, 7212, 7213, 7214, 7215, 7216, 7217, 7218, 7219, 7220, 7221, 7222,7223, 7224, 7225, 7226, 7227, 7228, 7229, 7230, 7231, 7232, 7233, 7234,7235, 7236, 7237, 7238, 7239, 7240, 7241, 7242, 7243, 7244, 7245, 7246,7247, 7248, 7249, 7250, 7251, 7252, 7253, 7254, 7255, 7256, 7257, 7258,7259, 7260, 7261, 7262, 7263, 7264, 7265, 7266, 7267, 7268, 7269, 7270,7271, 7272, 7273, 7274, 7275, 7276, 7277, 7278, 7279, 7280, 7281, 7282,7283, 7284, 7285, 7286, 7287, 7288, 7289, 7290, 7291, 7292, 7293, 7294,7295, 7296, 7297, 7298, 7299, 7300, 7301, 7302, 7303, 7304, 7305, 7306,7307, 7308, 7309, 7310, 7311, 7312, 7313, 7314, 7315, 7316, 7317, 7318,7319, 7320, 7321, 7322, 7323, 7324, 7325, 7326, 7327, 7328, 7329, 7330,7331, 7332, 7333, 7334, 7335, 7336, 7337, 7338, 7339, 7340, 7341, 7342,7343, 7344, 7345, 7346, 7347, 7348, 7349, 7350, 7351, 7352, 7353, 7354,7355, 7356, 7357, 7358, 7359, 7360, 7361, 7362, 7363, 7364, 7365, 7366,7367, 7368, 7369, 7370, 7371, 7372, 7373, 7374, 7375, 7376, 7377, 7378,7379, 7380, 7381, 7382, 7383, 7384, 7385, 7386, 7387, 7388, 7389, 7390,7391, 7392, 7393, 7394, 7395, 7396, 7397, 7398, 7399, 7400, 7401, 7402,7403, 7404, 7405, 7406, 7407, 7408, 7409, 7410, 7411, 7412, 7413, 7414,7415, 7416, 7417, 7418, 7419, 7420, 7421, 7422, 7423, 7424, 7425, 7426,7427, 7428, 7429, 7430, 7431, 7432, 7433, 7434, 7435, 7436, 7437, 7438,7439, 7440, 7441, 7442, 7443, 7444, 7445, 7446, 7447, 7448, 7449, 7450,7451, 7452, 7453, 7454, 7455, 7456, 7457, 7458, 7459, 7460, 7461, 7462,7463, 7464, 7465, 7466, 7467, 7468, 7469, 7470, 7471, 7472, 7473, 7474,7475, 7476, 7477, 7478, 7479, 7480, 7481, 7482, 7483, 7484, 7485, 7486,7487, 7488, 7489, 7490, 7491, 7492, 7493, 7494, 7495, 7496, 7497, 7498,7499, 7500, 7501, 7502, 7503, 7504, 7505, 7506, 7507, 7508, 7509, 7510,7511, 7512, 7513, 7514, 7515, 7516, 7517, 7518, 7519, 7520, 7521, 7522,7523, 7524, 7525, 7526, 7527, 7528, 7529, 7530, 7531, 7532, 7533, 7534,7535, 7536, 7537, 7538, 7539, 7540, 7541, 7542, 7543, 7544, 7545, 7546,7547, 7548, 7549, 7550, 7551, 7552, 7553, 7554, 7555, 7556, 7557, 7558,7559, 7560, 7561, 7562, 7563, 7564, 7565, 7566, 7567, 7568, 7569, 7570,7571, 7572, 7573, 7574, 7575, 7576, 7577, 7578, 7579, 7580, 7581, 7582,7583, 7584, 7585, 7586, 7587, 7588, 7589, 7590, 7591, 7592, 7593, 7594,7595, 7596, 7597, 7598, 7599, 7600, 7601, 7602, 7603, 7604, 7605, 7606,7607, 7608, 7609, 7610, 7611, 7612, 7613, 7614, 7615, 7616, 7617, 7618,7619, 7620, 7621, 7622, 7623, 7624, 7625, 7626, 7627, 7628, 7629, 7630,7631, 7632, 7633, 7634, 7635, 7636, 7637, 7638, 7639, 7640, 7641, 7642,7643, 7644, 7645, 7646, 7647, 7648, 7649, 7650, 7651, 7652, 7653, 7654,7655, 7656, 7657, 7658, 7659, 7660, 7661, 7662, 7663, 7664, 7665, 7666,7667, 7668, 7669, 7670, 7671, 7672, 7673, 7674, 7675, 7676, 7677, 7678,7679, 7680, 7681, 7682, 7683, 7684, 7685, 7686, 7687, 7688, 7689, 7690,7691, 7692, 7693, 7694, 7695, 7696, 7697, 7698, 7699, 7700, 7701, 7702,7703, 7704, 7705, 7706, 7707, 7708, 7709, 7710, 7711, 7712, 7713, 7714,7715, 7716, 7717, 7718, 7719, 7720, 7721, 7722, 7723, 7724, 7725, 7726,7727, 7728, 7729, 7730, 7731, 7732, 7733, 7734, 7735, 7736, 7737, 7738,7739, 7740, 7741, 7742, 7743, 7744, 7745, 7746, 7747, 7748, 7749, 7750,7751, 7752, 7753, 7754, 7755, 7756, 7757, 7758, 7759, 7760, 7761, 7762,7763, 7764, 7765, 7766, 7767, 7768, 7769, 7770, 7771, 7772, 7773, 7774,7775, 7776, 7777, 7778, 7779, 7780, 7781, 7782, 7783, 7784, 7785, 7786,7787, 7788, 7789, 7790, 7791, 7792, 7793, 7794, 7795, 7796, 7797, 7798,7799, 7800, 7801, 7802, 7803, 7804, 7805, 7806, 7807, 7808, 7809, 7810,7811, 7812, 7813, 7814, 7815, 7816, 7817, 7818, 7819, 7820, 7821, 7822,7823, 7824, 7825, 7826, 7827, 7828, 7829, 7830, 7831, 7832, 7833, 7834,7835, 7836, 7837, 7838, 7839, 7840, 7841, 7842, 7843, 7844, 7845, 7846,7847, 7848, 7849, 7850, 7851, 7852, 7853, 7854, 7855, 7856, 7857, 7858,7859, 7860, 7861, 7862, 7863, 7864, 7865, 7866, 7867, 7868, 7869, 7870,7871, 7872, 7873, 7874, 7875, 7876, 7877, 7878, 7879, 7880, 7881, 7882,7883, 7884, 7885, 7886, 7887, 7888, 7889, 7890, 7891, 7892, 7893, 7894,7895, 7896, 7897, 7898, 7899, 7900, 7901, 7902, 7903, 7904, 7905, 7906,7907, 7908, 7909, 7910, 7911, 7912, 7913, 7914, 7915, 7916, 7917, 7918,7919, 7920, 7921, 7922, 7923, 7924, 7925, 7926, 7927, 7928, 7929, 7930,7931, 7932, 7933, 7934, 7935, 7936, 7937, 7938, 7939, 7940, 7941, 7942,7943, 7944, 7945, 7946, 7947, 7948, 7949, 7950, 7951, 7952, 7953, 7954,7955, 7956, 7957, 7958, 7959, 7960, 7961, 7962, 7963, 7964, 7965, 7966,7967, 7968, 7969, 7970, 7971, 7972, 7973, 7974, 7975, 7976, 7977, 7978,7979, 7980, 7981, 7982, 7983, 7984, 7985, 7986, 7987, 7988, 7989, 7990,7991, 7992, 7993, 7994, 7995, 7996, 7997, 7998, 7999, 8000, 8001, 8002,8003, 8004, 8005, 8006, 8007, 8008, 8009, 8010, 8011, 8012, 8013, 8014,8015, 8016, 8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024, 8025, 8026,8027, 8028, 8029, 8030, 8031, 8032, 8033, 8034, 8035, 8036, 8037, 8038,8039, 8040, 8041, 8042, 8043, 8044, 8045, 8046, 8047, 8048, 8049, 8050,8051, 8052, 8053, 8054, 8055, 8056, 8057, 8058, 8059, 8060, 8061, 8062,8063, 8064, 8065, 8066, 8067, 8068, 8069, 8070, 8071, 8072, 8073, 8074,8075, 8076, 8077, 8078, 8079, 8080, 8081, 8082, 8083, 8084, 8085, 8086,8087, 8088, 8089, 8090, 8091, 8092, 8093, 8094, 8095, 8096, 8097, 8098,8099, 8100, 8101, 8102, 8103, 8104, 8105, 8106, 8107, 8108, 8109, 8110,8111, 8112, 8113, 8114, 8115, 8116, 8117, 8118, 8119, 8120, 8121, 8122,8123, 8124, 8125, 8126, 8127, 8128, 8129, 8130, 8131, 8132, 8133, 8134,8135, 8136, 8137, 8138, 8139, 8140, 8141, 8142, 8143, 8144, 8145, 8146,8147, 8148, 8149, 8150, 8151, 8152, 8153, 8154, 8155, 8156, 8157, 8158,8159, 8160, 8161, 8162, 8163, 8164, 8165, 8166, 8167, 8168, 8169, 8170,8171, 8172, 8173, 8174, 8175, 8176, 8177, 8178, 8179, 8180, 8181, 8182,8183, 8184, 8185, 8186, 8187, 8188, 8189, 8190, 8191, 8192, 8193, 8194,8195, 8196, 8197, 8198, 8199, 8200, 8201, 8202, 8203, 8204, 8205, 8206,8207, 8208, 8209, 8210, 8211, 8212, 8213, 8214, 8215, 8216, 8217, 8218,8219, 8220, 8221, 8222, 8223, 8224, 8225, 8226, 8227, 8228, 8229, 8230,8231, 8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241, 8242,8243, 8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251, 8252, 8253, 8254,8255, 8256, 8257, 8258, 8259, 8260, 8261, 8262, 8263, 8264, 8265, 8266,8267, 8268, 8269, 8270, 8271, 8272, 8273, 8274, 8275, 8276, 8277, 8278,8279, 8280, 8281, 8282, 8283, 8284, 8285, 8286, 8287, 8288, 8289, 8290,8291, 8292, 8293, 8294, 8295, 8296, 8297, 8298, 8299, 8300, 8301, 8302,8303, 8304, 8305, 8306, 8307, 8308, 8309, 8310, 8311, 8312, 8313, 8314,8315, 8316, 8317, 8318, 8319, 8320, 8321, 8322, 8323, 8324, 8325, 8326,8327, 8328, 8329, 8330, 8331, 8332, 8333, 8334, 8335, 8336, 8337, 8338,8339, 8340, 8341, 8342, 8343, 8344, 8345, 8346, 8347, 8348, 8349, 8350,8351, 8352, 8353, 8354, 8355, 8356, 8357, 8358, 8359, 8360, 8361, 8362,8363, 8364, 8365, 8366, 8367, 8368, 8369, 8370, 8371, 8372, 8373, 8374,8375, 8376, 8377, 8378, 8379, 8380, 8381, 8382, 8383, 8384, 8385, 8386,8387, 8388, 8389, 8390, 8391, 8392, 8393, 8394, 8395, 8396, 8397, 8398,8399, 8400, 8401, 8402, 8403, 8404, 8405, 8406, 8407, 8408, 8409, 8410,8411, 8412, 8413, 8414, 8415, 8416, 8417, 8418, 8419, 8420, 8421, 8422,8423, 8424, 8425, 8426, 8427, 8428, 8429, 8430, 8431, 8432, 8433, 8434,8435, 8436, 8437, 8438, 8439, 8440, 8441, 8442, 8443, 8444, 8445, 8446,8447, 8448, 8449, 8450, 8451, 8452, 8453, 8454, 8455, 8456, 8457, 8458,8459, 8460, 8461, 8462, 8463, 8464, 8465, 8466, 8467, 8468, 8469, 8470,8471, 8472, 8473, 8474, 8475, 8476, 8477, 8478, 8479, 8480, 8481, 8482,8483, 8484, 8485, 8486, 8487, 8488, 8489, 8490, 8491, 8492, 8493, 8494,8495, 8496, 8497, 8498, 8499, 8500, 8501, 8502, 8503, 8504, 8505, 8506,8507, 8508, 8509, 8510, 8511, 8512, 8513, 8514, 8515, 8516, 8517, 8518,8519, 8520, 8521, 8522, 8523, 8524, 8525, 8526, 8527, 8528, 8529, 8530,8531, 8532, 8533, 8534, 8535, 8536, 8537, 8538, 8539, 8540, 8541, 8542,8543, 8544, 8545, 8546, 8547, 8548, 8549, 8550, 8551, 8552, 8553, 8554,8555, 8556, 8557, 8558, 8559, 8560, 8561, 8562, 8563, 8564, 8565, 8566,8567, 8568, 8569, 8570, 8571, 8572, 8573, 8574, 8575, 8576, 8577, 8578,8579, 8580, 8581, 8582, 8583, 8584, 8585, 8586, 8587, 8588, 8589, 8590,8591, 8592, 8593, 8594, 8595, 8596, 8597, 8598, 8599, 8600, 8601, 8602,8603, 8604, 8605, 8606, 8607, 8608, 8609, 8610, 8611, 8612, 8613, 8614,8615, 8616, 8617, 8618, 8619, 8620, 8621, 8622, 8623, 8624, 8625, 8626,8627, 8628, 8629, 8630, 8631, 8632, 8633, 8634, 8635, 8636, 8637, 8638,8639, 8640, 8641, 8642, 8643, 8644, 8645, 8646, 8647, 8648, 8649, 8650,8651, 8652, 8653, 8654, 8655, 8656, 8657, 8658, 8659, 8660, 8661, 8662,8663, 8664, 8665, 8666, 8667, 8668, 8669, 8670, 8671, 8672, 8673, 8674,8675, 8676, 8677, 8678, 8679, 8680, 8681, 8682, 8683, 8684, 8685, 8686,8687, 8688, 8689, 8690, 8691, 8692, 8693, 8694, 8695, 8696, 8697, 8698,8699, 8700, 8701, 8702, 8703, 8704, 8705, 8706, 8707, 8708, 8709, 8710,8711, 8712, 8713, 8714, 8715, 8716, 8717, 8718, 8719, 8720, 8721, 8722,8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730, 8731, 8732, 8733, 8734,8735, 8736, 8737, 8738, 8739, 8740, 8741, 8742, 8743, 8744, 8745, 8746,8747, 8748, 8749, 8750, 8751, 8752, 8753, 8754, 8755, 8756, 8757, 8758,8759, 8760, 8761, 8762, 8763, 8764, 8765, 8766, 8767, 8768, 8769, 8770,8771, 8772, 8773, 8774, 8775, 8776, 8777, 8778, 8779, 8780, 8781, 8782,8783, 8784, 8785, 8786, 8787, 8788, 8789, 8790, 8791, 8792, 8793, 8794,8795, 8796, 8797, 8798, 8799, 8800, 8801, 8802, 8803, 8804, 8805, 8806,8807, 8808, 8809, 8810, 8811, 8812, 8813, 8814, 8815, 8816, 8817, 8818,8819, 8820, 8821, 8822, 8823, 8824, 8825, 8826, 8827, 8828, 8829, 8830,8831, 8832, 8833, 8834, 8835, 8836, 8837, 8838, 8839, 8840, 8841, 8842,8843, 8844, 8845, 8846, 8847, 8848, 8849, 8850, 8851, 8852, 8853, 8854,8855, 8856, 8857, 8858, 8859, 8860, 8861, 8862, 8863, 8864, 8865, 8866,8867, 8868, 8869, 8870, 8871, 8872, 8873, 8874, 8875, 8876, 8877, 8878,8879, 8880, 8881, 8882, 8883, 8884, 8885, 8886, 8887, 8888, 8889, 8890,8891, 8892, 8893, 8894, 8895, 8896, 8897, 8898, 8899, 8900, 8901, 8902,8903, 8904, 8905, 8906, 8907, 8908, 8909, 8910, 8911, 8912, 8913, 8914,8915, 8916, 8917, 8918, 8919, 8920, 8921, 8922, 8923, 8924, 8925, 8926,8927, 8928, 8929, 8930, 8931, 8932, 8933, 8934, 8935, 8936, 8937, 8938,8939, 8940, 8941, 8942, 8943, 8944, 8945, 8946, 8947, 8948, 8949, 8950,8951, 8952, 8953, 8954, 8955, 8956, 8957, 8958, 8959, 8960, 8961, 8962,8963, 8964, 8965, 8966, 8967, 8968, 8969, 8970, 8971, 8972, 8973, 8974,8975, 8976, 8977, 8978, 8979, 8980, 8981, 8982, 8983, 8984, 8985, 8986,8987, 8988, 8989, 8990, 8991, 8992, 8993, 8994, 8995, 8996, 8997, 8998,8999, 9000, 9001, 9002, 9003, 9004, 9005, 9006, 9007, 9008, 9009, 9010,9011, 9012, 9013, 9014, 9015, 9016, 9017, 9018, 9019, 9020, 9021, 9022,9023, 9024, 9025, 9026, 9027, 9028, 9029, 9030, 9031, 9032, 9033, 9034,9035, 9036, 9037, 9038, 9039, 9040, 9041, 9042, 9043, 9044, 9045, 9046,9047, 9048, 9049, 9050, 9051, 9052, 9053, 9054, 9055, 9056, 9057, 9058,9059, 9060, 9061, 9062, 9063, 9064, 9065, 9066, 9067, 9068, 9069, 9070,9071, 9072, 9073, 9074, 9075, 9076, 9077, 9078, 9079, 9080, 9081, 9082,9083, 9084, 9085, 9086, 9087, 9088, 9089, 9090, 9091, 9092, 9093, 9094,9095, 9096, 9097, 9098, 9099, 9100, 9101, 9102, 9103, 9104, 9105, 9106,9107, 9108, 9109, 9110, 9111, 9112, 9113, 9114, 9115, 9116, 9117, 9118,9119, 9120, 9121, 9122, 9123, 9124, 9125, 9126, 9127, 9128, 9129, 9130,9131, 9132, 9133, 9134, 9135, 9136, 9137, 9138, 9139, 9140, 9141, 9142,9143, 9144, 9145, 9146, 9147, 9148, 9149, 9150, 9151, 9152, 9153, 9154,9155, 9156, 9157, 9158, 9159, 9160, 9161, 9162, 9163, 9164, 9165, 9166,9167, 9168, 9169, 9170, 9171, 9172, 9173, 9174, 9175, 9176, 9177, 9178,9179, 9180, 9181, 9182, 9183, 9184, 9185, 9186, 9187, 9188, 9189, 9190,9191, 9192, 9193, 9194, 9195, 9196, 9197, 9198, 9199, 9200, 9201, 9202,9203.

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more peptides, as determined bycomparing the sequences. In the art, identity also means the degree ofsequence relatedness between peptides, as determined by the number ofmatches between strings of two or more amino acid residues. Identitymeasures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated peptides can be readily calculated by known methods. Suchmethods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

In some embodiments, the polypeptide variant may have the same or asimilar activity as the reference oncology-related polypeptide.Alternatively, the variant may have an altered activity (e.g., increasedor decreased) relative to a reference oncology-related polypeptide.Generally, variants of a particular oncology-related polynucleotide oroncology-related polypeptide of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity tothat particular reference oncology-related polynucleotide oroncology-related polypeptide as determined by sequence alignmentprograms and parameters described herein and known to those skilled inthe art. Such tools for alignment include those of the BLAST suite(Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schäffer, JinghuiZhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “GappedBLAST and PSI-BLAST: a new generation of protein database searchprograms”, Nucleic Acids Res. 25:3389-3402.) Other tools are describedherein, specifically in the definition of “identity.”

Default parameters in the BLAST algorithm include, for example, anexpect threshold of 10, Word size of 28, Match/Mismatch Scores 1, −2,Gap costs Linear. Any filter can be applied as well as a selection forspecies specific repeats, e.g., Homo sapiens.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs and/or oncology-related mmRNA may beused to treat a disease, disorder and/or condition in a subject.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs and/or oncology-related mmRNA may beused to reduce, eliminate or prevent tumor growth in a subject.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs and/or oncology-related mmRNA may beused to recude and/or ameliorate at least one symptom of cancer in asubject. A symptom of cancer may include, but is not limited to,weakness, aches and pains, fever, fatigue, weight loss, blood clots,increased blood calcium levels, low white blood cell count, short ofbreath, dizziness, headaches, hyperpigmentation, jaundice, erthema,pruritis, excessive hair growth, change in bowel habits, change inbladder function, long-lasting sores, white patches inside the mouth,white spots on the tongue, unusual bleeding or discharge, thickening orlump on parts of the body, indigestion, trouble swallowing, changes inwarts or moles, change in new skin and nagging cough or hoarseness.Further, the oncology-related polynucleotides, oncology-related primaryconstructs and/or oncology-related mmRNA may reduce a side-effectassociated with cancer such as, but not limited to, chemo brain,peripheral neuropathy, fatigue, depression, nausea, vomiting, pain,anemia, lymphedema, infections, sexual side effects, reduced fertilityor infertility, ostomics, insomnia and hair loss.

Oncology-Related Proteins or Oncology-Related Peptides

The oncology-related primary constructs or oncology-related mmRNAdisclosed herein, may encode one or more validated or “in testing”oncology-related proteins or oncology-related peptides.

According to the present invention, one or more oncology-relatedproteins or oncology-related peptides currently being marketed or indevelopment may be encoded by the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention. While not wishing to be bound by theory, it isbelieved that incorporation into the oncology-related primary constructsor oncology-related mmRNA of the invention will result in improvedtherapeutic efficacy due at least in part to the specificity, purity andselectivity of the construct designs.

The oncology-related polynucleotides, oncology-related primaryconstructs and/or oncology-related mmRNA may alter a biological and/orphysiolocial process and/or compound such as, but not limited to, thecell cycle, the DNA damage response (e.g., DNA damage repair),apoptosis, angiogenesis, cell motility, the epithelial to mesenchymaltransition in epithelial cells, the phosphatidyl inositol 3 (PI3)kinase/Akt cellular signaling pathway, telomerase activity and/orexpression, tumor metastasis, tumorigenesis, cathepsins, cellsenescence, receptor tyrosine kinase signaling, metabolism and drugmetabolism, G protein signaling, growth factors and receptors, heatshock proteins, histone deacetylases, hormone receptors, hypoxia, polyADP-ribose polymerases, protein kinases, RAS signaling, topisomerases,transcription factors and tumor suppressor activity in cancerous,precancerous and/or other cells.

In one embodiment, the polynucleotides, oncology-related primaryconstructs and/or oncology-related mmRNA may used to express apolypeptide in cells or tissues for the purpose of replacing the proteinproduced from a deleted or mutated gene.

Further, the oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA of the invention may be used totreat cancer which has been caused by carcinogens of natural and/orsynthetic origin. In another embodiment, the use of the oncology-relatedpolynucleotides, oncology-related primary constructs and/oroncology-related mmRNA may be used to treat cancer caused by otherorganisms and/or cancers caused by viral infection.

Flanking Regions: Untranslated Regions (UTRs)

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon;whereas, the 3′UTR starts immediately following the stop codon andcontinues until the transcriptional termination signal. There is growingbody of evidence about the regulatory roles played by the UTRs in termsof stability of the nucleic acid molecule and translation. Theregulatory features of a UTR can be incorporated into theoncology-related polynucleotides, oncology-related primary constructsand/or oncology-related mmRNA of the present invention to enhance thestability of the molecule. The specific features can also beincorporated to ensure controlled down-regulation of the transcript incase they are misdirected to undesired organs sites.

5′ UTR and Translation Initiation

Natural 5′UTRs bear features which play roles in for translationinitiation. They harbor signatures like Kozak sequences which arecommonly known to be involved in the process by which the ribosomeinitiates translation of many genes. Kozak sequences have the consensusCCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three basesupstream of the start codon (AUG), which is followed by another ‘G’.5′UTR also have been known to form secondary structures which areinvolved in elongation factor binding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andoncology-related protein production of the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA of the invention. For example, introduction of 5′ UTR ofliver-expressed mRNA, such as albumin, serum amyloid A, ApolipoproteinAB/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII,could be used to enhance expression of a nucleic acid molecule, such asa mmRNA, in hepatic cell lines or liver. Likewise, use of 5′ UTR fromother tissue-specific mRNA to improve expression in that tissue ispossible—for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), forendothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF,GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), foradipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lungepithelial cells (SP-A/B/C/D).

Other non-UTR sequences may be incorporated into the 5′ (or 3′ UTR)UTRs. For example, introns or portions of introns sequences may beincorporated into the flanking regions of the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA of the invention. Incorporation of intronic sequences may increaseprotein production as well as mRNA levels.

3′ UTR and the AU Rich Elements

3′UTRs are known to have stretches of Adenosines and Uridines embeddedin them. These AU rich signatures are particularly prevalent in geneswith high rates of turnover. Based on their sequence features andfunctional properties, the AU rich elements (AREs) can be separated intothree classes (Chen et al, 1995): Class I AREs contain several dispersedcopies of an AUUUA motif within U-rich regions. C-Myc and MyoD containclass I AREs. Class II AREs possess two or more overlappingUUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREsinclude GM-CSF and TNF-α. Class III ARES are less well defined. These Urich regions do not contain an AUUUA motif c-Jun and Myogenin are twowell-studied examples of this class. Most proteins binding to the AREsare known to destabilize the messenger, whereas members of the ELAVfamily, most notably HuR, have been documented to increase the stabilityof mRNA. HuR binds to AREs of all the three classes. Engineering the HuRspecific binding sites into the 3′ UTR of nucleic acid molecules willlead to HuR binding and thus, stabilization of the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA of the invention. When engineering specific oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA, one or more copies of an ARE can be introduced to makeoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA of the invention less stable and thereby curtailtranslation and decrease production of the resultant protein. Likewise,AREs can be identified and removed or mutated to increase theintracellular stability and thus increase translation and production ofthe resultant protein. Transfection experiments can be conducted inrelevant cell lines, using oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of theinvention and protein production can be assayed at various time pointspost-transfection. For example, cells can be transfected with differentARE-engineering molecules and by using an ELISA kit to the relevantprotein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48hour, and 7 days post-transfection.

Incorporating microRNA Binding Sites

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′UTR of nucleic acid molecules and down-regulate gene expressioneither by reducing nucleic acid molecule stability or by inhibitingtranslation. The oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA of the invention maycomprise one or more microRNA target sequences, microRNA sequences, ormicroRNA seeds. Such sequences may correspond to any known microRNA suchas those taught in US Publication US2005/0261218 and US PublicationUS2005/0059005, the contents of which are incorporated herein byreference in their entirety.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked by an adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105; each of which is hereinincorporated by reference in their entirety. The bases of the microRNAseed have complete complementarity with the target sequence. Byengineering microRNA target sequences into the 3′UTR of oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA of the invention one can target the molecule for degradation orreduced translation, provided the microRNA in question is available.This process will reduce the hazard of off target effects upon nucleicacid molecule delivery. Identification of microRNA, microRNA targetregions, and their expression patterns and role in biology have beenreported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand andCheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia2012 26:404-413 (2011 Dec. 20. doi: 10.1038/leu.2011.356); Bartel Cell2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; each ofwhich is herein incorporated by reference in their entirety).

For example, if the nucleic acid molecule is an mRNA and is not intendedto be delivered to the liver but ends up there, then miR-122, a microRNAabundant in liver, can inhibit the expression of the gene of interest ifone or multiple target sites of miR-122 are engineered into the 3′UTR ofthe oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA. Introduction of one or multiplebinding sites for different microRNA can be engineered to furtherdecrease the longevity, stability, and protein translation ofoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA. As used herein, the term “microRNA site” refersto a microRNA target site or a microRNA recognition site, or anynucleotide sequence to which a microRNA binds or associates. It shouldbe understood that “binding” may follow traditional Watson-Crickhybridization rules or may reflect any stable association of themicroRNA with the target sequence at or adjacent to the microRNA site.

Conversely, for the purposes of the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention, microRNA binding sites can be engineered out of (i.e.removed from) sequences in which they naturally occur in order toincrease protein expression in specific tissues. For example, miR-122binding sites may be removed to improve protein expression in the liver.Regulation of expression in multiple tissues can be accomplished throughintroduction or removal or one or several microRNA binding sites.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulatecomplex biological processes such as angiogenesis (miR-132) (Anand andCheresh Curr Opin Hematol 2011 18:171-176; herein incorporated byreference in its entirety). In the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention, binding sites for microRNAs that are involved in suchprocesses may be removed or introduced, in order to tailor theexpression of the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA expression to biologicallyrelevant cell types or to the context of relevant biological processes.A listing of MicroRNA, miR sequences and miR binding sites is listed inTable 9 of U.S. Provisional Application No. 61/753,661 filed Jan. 17,2013, in Table 9 of U.S. Provisional Application No. 61/754,159 filedJan. 18, 2013, and in Table 7 of U.S. Provisional Application No.61/758,921 filed Jan. 31, 2013, each of which are herein incorporated byreference in their entireties.

Lastly, through an understanding of the expression patterns of microRNAin different oncology-related cell types, oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA can be engineered for more targeted expression in specific celltypes or only under specific biological conditions. Through introductionof tissue-specific microRNA binding sites, oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA could be designed that would be optimal for oncology-relatedprotein expression in a tissue or in the context of a biologicalcondition. Examples of use of microRNA to drive tissue ordisease-specific gene expression are listed (Getner and Naldini, TissueAntigens. 2012, 80:393-403; herein incorporated by reference in itsentirety). In addition, microRNA seed sites can be incorporated intomRNA to decrease expression in certain cells which results in abiological improvement. An example of this is incorporation of miR-142sites into a UGT1A1-expressing lentiviral vector. The presence ofmiR-142 seed sites reduced expression in hematopoietic cells, and as aconsequence reduced expression in antigen-presentating cells, leading tothe absence of an immune response against the virally expressed UGT1A1(Schmitt et al., Gastroenterology 2010; 139:999-1007;Gonzalez-Asequinolaza et al. Gastroenterology 2010, 139:726-729; each ofwhich are herein incorporated by reference in their entirety).Incorporation of miR-142 sites into modified mRNA could not only reduceexpression of the encoded protein in hematopoietic cells, but could alsoreduce or abolish immune responses to the mRNA-encoded protein.Incorporation of miR-142 seed sites (one or multiple) into mRNA would beimportant in the case of treatment of patients with complete proteindeficiencies such as, but not limited to, UGT1A1 type I, LDLR-deficientpatients abd CRIM-negative Pompe patients.

Transfection experiments can be conducted in relevant cell lines, usingengineered oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA and protein production can beassayed at various time points post-transfection. For example, cells canbe transfected with different microRNA binding site-engineeringoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA and by using an ELISA kit to the relevant proteinand assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, 72hour and 7 days post-transfection. In vivo experiments can also beconducted using microRNA-binding site-engineered molecules to examinechanges in tissue-specific expression of formulated oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA.

5′ Capping

The 5′ cap structure of an mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

Modifications to the oncology-related polynucleotides, oncology-relatedprimary constructs, and oncology-related mmRNA of the present inventionmay generate a non-hydrolyzable cap structure preventing decapping andthus increasing mRNA half-life. Because cap structure hydrolysisrequires cleavage of 5′-ppp-5′ phosphorodiester linkages, modifiednucleotides may be used during the capping reaction. For example, aVaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may beused with α-thio-guanosine nucleotides according to the manufacturer'sinstructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap.Additional modified guanosine nucleotides may be used such asα-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the mRNA (as mentioned above) on the2′-hydroxyl group of the sugar ring. Multiple distinct 5′-cap structurescan be used to generate the 5′-cap of a nucleic acid molecule, such asan mRNA molecule.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e. endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e. non-enzymatically) orenzymatically synthesized and/or linked to a nucleic acid molecule.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′mppp-G;which may equivalently be designated 3′ O-Me-m7G(5)ppp(5′)G). The 3′-Oatom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA).The N7- and 3′-O-methylated guanine provides the terminal moiety of thecapped nucleic acid molecule (e.g. mRNA or mmRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

While cap analogs allow for the concomitant capping of a nucleic acidmolecule in an in vitro transcription reaction, up to 20% of transcriptscan remain uncapped. This, as well as the structural differences of acap analog from an endogenous 5′-cap structures of nucleic acidsproduced by the endogenous, cellular transcription machinery, may leadto reduced translational competency and reduced cellular stability.

Oncology-related polynucleotides, oncology-related primary constructsand oncology-related mmRNA of the invention may also be cappedpost-transcriptionally, using enzymes, in order to generate moreauthentic 5′-cap structures. As used herein, the phrase “more authentic”refers to a feature that closely mirrors or mimics, either structurallyor functionally, an endogenous or wild type feature. That is, a “moreauthentic” feature is better representative of an endogenous, wild-type,natural or physiological cellular function and/or structure as comparedto synthetic features or analogs, etc., of the prior art, or whichoutperforms the corresponding endogenous, wild-type, natural orphysiological feature in one or more respects. Non-limiting examples ofmore authentic 5′cap structures of the present invention are thosewhich, among other things, have enhanced binding of cap bindingproteins, increased half life, reduced susceptibility to 5′endonucleases and/or reduced 5′decapping, as compared to synthetic 5′capstructures known in the art (or to a wild-type, natural or physiological5′cap structure). For example, recombinant Vaccinia Virus Capping Enzymeand recombinant 2′-O-methyltransferase enzyme can create a canonical5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNAand a guanine cap nucleotide wherein the cap guanine contains an N7methylation and the 5′-terminal nucleotide of the mRNA contains a2′-O-methyl. Such a structure is termed the Cap1 structure. This capresults in a higher translational-competency and cellular stability anda reduced activation of cellular pro-inflammatory cytokines, ascompared, e.g., to other 5′cap analog structures known in the art. Capstructures include, but are not limited to, 7mG(5′)ppp(5′)N,pN2p (cap0), 7mG(5′)ppp(5′)N1mpNp (cap 1), and 7mG(5′)-ppp(5′)N1mpN2mp (cap 2).

Because the oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA may be cappedpost-transcriptionally, and because this process is more efficient,nearly 100% of the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA may be capped. This is incontrast to ˜80% when a cap analog is linked to an mRNA in the course ofan in vitro transcription reaction.

According to the present invention, 5′ terminal caps may includeendogenous caps or cap analogs. According to the present invention, a 5′terminal cap may comprise a guanine analog. Useful guanine analogsinclude, but are not limited to, inosine, N1-methyl-guanosine,2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Viral Sequences

Additional viral sequences such as, but not limited to, the translationenhancer sequence of the barley yellow dwarf virus (BYDV-PAV), theJaagsiekte sheep retrovirus (JSRV) and/or the Enzootic nasal tumor virus(See e.g., International Pub. No. WO2012129648; herein incorporated byreference in its entirety) can be engineered and inserted in the 3′ UTRof the oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA of the invention and can stimulatethe translation of the construct in vitro and in vivo. Transfectionexperiments can be conducted in relevant cell lines at and proteinproduction can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day7 post-transfection.

IRES Sequences

Further, provided are oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA which may contain aninternal ribosome entry site (IRES). First identified as a featurePicorna virus RNA, IRES plays an important role in initiating proteinsynthesis in absence of the 5′ cap structure. An IRES may act as thesole ribosome binding site, or may serve as one of multiple ribosomebinding sites of an mRNA. Oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA containingmore than one functional ribosome binding site may encode severaloncology-related peptides or oncology-related polypeptides that aretranslated independently by the ribosomes (“multicistronic nucleic acidmolecules”). When oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA are provided with an IRES,further optionally provided is a second translatable region. Examples ofIRES sequences that can be used according to the invention includewithout limitation, those from picornaviruses (e.g. FMDV), pest viruses(CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV),foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV),classical swine fever viruses (CSFV), murine leukemia virus (MLV),simian immune deficiency viruses (SIV) or cricket paralysis viruses(CrPV).

Poly-A Tails

During RNA processing, a long chain of adenine nucleotides (poly-A tail)may be added to a polynucleotide such as an mRNA molecule in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript may be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between, for example,approximately 100 and 250 residues long.

It has been discovered that unique poly-A tail lengths provide certainadvantages to the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA of the present invention.

Generally, the length of a poly-A tail of the present invention isgreater than 30 nucleotides in length. In another embodiment, the poly-Atail is greater than 35 nucleotides in length (e.g., at least or greaterthan about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500,and 3,000 nucleotides). In some embodiments, the oncology-relatedpolynucleotide, oncology-related primary construct, or oncology-relatedmmRNA includes from about 30 to about 3,000 nucleotides (e.g., from 30to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750,from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500,from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000,from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000,from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from2,000 to 2,500, and from 2,500 to 3,000).

In one embodiment, the poly-A tail is designed relative to the length ofthe overall oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA. This design may be based on thelength of the coding region, the length of a particular feature orregion (such as the first or flanking regions), or based on the lengthof the ultimate product expressed from the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA.

In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA or featurethereof. The poly-A tail may also be designed as a fraction ofoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA to which it belongs. In this context, the poly-Atail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the totallength of the construct or the total length of the construct minus thepoly-A tail. Further, engineered binding sites and conjugation ofoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA for Poly-A binding protein may enhanceexpression.

Additionally, multiple distinct oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA may belinked together to the PABP (Poly-A binding protein) through the 3′-endusing modified nucleotides at the 3′-terminus of the poly-A tail.Transfection experiments can be conducted in relevant cell lines andoncology-related protein production can be assayed by ELISA at 12 hr, 24hr, 48 hr, 72 hr and day 7 post-transfection.

In one embodiment, the oncology-related polynucleotide andoncology-related primary constructs of the present invention aredesigned to include a polyA-G Quartet. The G-quartet is a cyclichydrogen bonded array of four guanine nucleotides that can be formed byG-rich sequences in both DNA and RNA. In this embodiment, the G-quartetis incorporated at the end of the poly-A tail. The resultantoncology-related mmRNA construct is assayed for stability, proteinproduction and other parameters including half-life at various timepoints. It has been discovered that the polyA-G quartet results inprotein production equivalent to at least 75% of that seen using apoly-A tail of 120 nucleotides alone.

Quantification

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be quantified in exosomes derived from one or morebodily fluid. As used herein “bodily fluids” include peripheral blood,serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood.Alternatively, exosomes may be retrieved from an organ selected from thegroup consisting of lung, heart, pancreas, stomach, intestine, bladder,kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus,liver, and placenta.

In the quantification method, a sample of not more than 2 mL is obtainedfrom the subject and the exosomes isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.In the analysis, the level or concentration of an oncology-relatedpolynucleotide, oncology-related primary construct or oncology-relatedmmRNA may be an expression level, presence, absence, truncation oralteration of the administered construct. It is advantageous tocorrelate the level with one or more clinical phenotypes or with anassay for a human disease biomarker. The assay may be performed usingconstruct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flowcytometry, electrophoresis, mass spectrometry, or combinations thereofwhile the exosomes may be isolated using immunohistochemical methodssuch as enzyme linked immunosorbent assay (ELISA) methods. Exosomes mayalso be isolated by size exclusion chromatography, density gradientcentrifugation, differential centrifugation, nanomembraneultrafiltration, immunoabsorbent capture, affinity purification,microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA remaining or delivered.This is possible because the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention differ from the endogenous forms due to the structuraland/or chemical modifications.

II. DESIGN AND SYNTHESIS OF MMRNA

Oncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA for use in accordance with the invention may beprepared according to any available technique including, but not limitedto chemical synthesis, enzymatic synthesis, which is generally termed invitro transcription (IVT) or enzymatic or chemical cleavage of a longerprecursor, etc. Methods of synthesizing RNAs are known in the art (see,e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach,Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P.(ed.) Oligonucleotide synthesis: methods and applications, Methods inMolecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press,2005; both of which are incorporated herein by reference).

The process of design and synthesis of the oncology-related primaryconstructs of the invention generally includes the steps of geneconstruction, mRNA production (either with or without modifications) andpurification. In the enzymatic synthesis method, a targetoncology-related polynucleotide sequence encoding the oncology-relatedpolypeptide of interest is first selected for incorporation into avector which will be amplified to produce a cDNA template. Optionally,the target oncology-related polynucleotide sequence and/or any flankingsequences may be codon optimized. The cDNA template is then used toproduce mRNA through in vitro transcription (IVT). After production, themRNA may undergo purification and clean-up processes. The steps of whichare provided in more detail below.

Gene Construction

The step of gene construction may include, but is not limited to genesynthesis, vector amplification, plasmid purification, plasmidlinearization and clean-up, and cDNA template synthesis and clean-up.

Gene Synthesis

Once an oncology-related polypeptide of interest, or target, is selectedfor production, an oncology-related primary construct is designed.Within the oncology-related primary construct, a first region of linkednucleosides encoding the polypeptide of interest may be constructedusing an open reading frame (ORF) of a selected nucleic acid (DNA orRNA) transcript. The ORF may comprise the wild type ORF, an isoform,variant or a fragment thereof. As used herein, an “open reading frame”or “ORF” is meant to refer to a nucleic acid sequence (DNA or RNA) whichis capable of encoding an oncology-related polypeptide of interest. ORFsoften begin with the start codon, ATG and end with a nonsense ortermination codon or signal.

Further, the nucleotide sequence of the first region may be codonoptimized. Codon optimization methods are known in the art and may beuseful in efforts to achieve one or more of several goals. These goalsinclude to match codon frequencies in target and host organisms toensure proper folding, bias GC content to increase mRNA stability orreduce secondary structures, minimize tandem repeat codons or base runsthat may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeprotein trafficking sequences, remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, to adjusttranslational rates to allow the various domains of the protein to foldproperly, or to reduce or eliminate problem secondary structures withinthe mRNA. Codon optimization tools, algorithms and services are known inthe art, non-limiting examples include services from GeneArt (LifeTechnologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. Inone embodiment, the ORF sequence is optimized using optimizationalgorithms. Codon options for each amino acid are given in Table 1.

TABLE 1 Codon Options Single Letter Amino Acid Code Codon OptionsIsoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA, CTG, TTA, TTG ValineV GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC Methionine M ATG CysteineC TGT, TGC Alanine A GCT, GCC, GCA, GCG Glycine G GGT, GGC, GGA, GGGProline P CCT, CCC, CCA, CCG Threonine T ACT, ACC, ACA, ACG Serine STCT, TCC, TCA, TCG, AGT, AGC Tyrosine Y TAT, TAC Tryptophan W TGGGlutamine Q CAA, CAG Asparagine N AAT, AAC Histidine H CAT, CAC Glutamicacid E GAA, GAG Aspartic acid D GAT, GAC Lysine K AAA, AAG Arginine RCGT, CGC, CGA, CGG, AGA, AGG Selenocysteine Sec UGA in mRNA in presenceof Selenocystein insertion element (SECIS) Stop codons Stop TAA, TAG,TGA

Features, which may be considered beneficial in some embodiments of thepresent invention, may be encoded by the oncology-related primaryconstruct and may flank the ORF as a first or second flanking region.The flanking regions may be incorporated into the oncology-relatedprimary construct before and/or after optimization of the ORF. It is notrequired that an oncology-related primary construct contain both a 5′and 3′ flanking region. Examples of such features include, but are notlimited to, untranslated regions (UTRs), Kozak sequences, an oligo(dT)sequence, and detectable tags and may include multiple cloning siteswhich may have XbaI recognition.

In some embodiments, a 5′ UTR and/or a 3′ UTR may be provided asflanking regions. Multiple 5′ or 3′ UTRs may be included in the flankingregions and may be the same or of different sequences. Any portion ofthe flanking regions, including none, may be codon optimized and any mayindependently contain one or more different structural or chemicalmodifications, before and/or after codon optimization. Combinations offeatures may be included in the first and second flanking regions andmay be contained within other features. For example, the ORF may beflanked by a 5′ UTR which may contain a strong Kozak translationalinitiation signal and/or a 3′ UTR which may include an oligo(dT)sequence for templated addition of a poly-A tail. The 5′UTR may comprisea first polynucleotide fragment and a second polynucleotide fragmentfrom the same and/or different genes such as the 5′UTRs described in USPatent Application Publication No. 20100293625, herein incorporated byreference in its entirety.

Tables 2 and 3 provide a listing of exemplary UTRs which may be utilizedin the oncology-related primary construct of the present invention asflanking regions. Shown in Table 2 is a listing of a 5′-untranslatedregion of the invention. Variants of 5′ UTRs may be utilized wherein oneor more nucleotides are added or removed to the termini, including A, T,C or G.

TABLE 2 5′-Untranslated Regions 5′ SEQ UTR Name/ ID IdentifierDescription Sequence NO. 5UTR- Upstream UTRGGGAAATAAGAGAGAAAAGAAGAGTAAGAAG 1 001 AAATATAAGAGCCACC 5UTR-Upstream UTR GGGAGATCAGAGAGAAAAGAAGAGTAAGAAG 2 002 AAATATAAGAGCCACC5UTR- Upstream UTR GGAATAAAAGTCTCAACACAACATATACAAAA 3 003CAAACGAATCTCAAGCAATCAAGCATTCTACT TCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTT ACGAACGATAGCAAC 5UTR- Upstream UTRGGGAGACAAGCUUGGCAUUCCGGUACUGUUG 4 004 GUAAAGCCACC

Shown in Table 3 is a representative listing of 3′-untranslated regionsof the invention. Variants of 3′ UTRs may be utilized wherein one ormore nucleotides are added or removed to the termini, including A, T, Cor G.

TABLE 3 3′-Untranslated Regions SEQ 3′UTR Name/ ID IdentifierDescription Sequence NO.  3UTR- Creatine GCGCCTGCCCACCTGCCACCGACTGCTGGAA 5 001 Kinase CCCAGCCAGTGGGAGGGCCTGGCCCACCAGAGTCCTGCTCCCTCACTCCTCGCCCCGCCCCCT GTCCCAGAGTCCCACCTGGGGGCTCTCTCCACCCTTCTCAGAGTTCCAGTTTCAACCAGAGTT CCAACCAATGGGCTCCATCCTCTGGATTCTGGCCAATGAAATATCTCCCTGGCAGGGTCCTC TTCTTTTCCCAGAGCTCCACCCCAACCAGGAGCTCTAGTTAATGGAGAGCTCCCAGCACACT CGGAGCTTGTGCTTTGTCTCCACGCAAAGCGATAAATAAAAGCATTGGTGGCCTTTGGTCTT TGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-Myoglobin GCCCCTGCCGCTCCCACCCCCACCCATCTGG  6 002GCCCCGGGTTCAAGAGAGAGCGGGGTCTGAT CTCGTGTAGCCATATAGAGTTTGCTTCTGAGTGTCTGCTTTGTTTAGTAGAGGTGGGCAGGAG GAGCTGAGGGGCTGGGGCTGGGGTGTTGAAGTTGGCTTTGCATGCCCAGCGATGCGCCTCC CTGTGGGATGTCATCACCCTGGGAACCGGGAGTGGCCCTTGGCTCACTGTGTTCTGCATGGTT TGGATCTGAATTAATTGTCCTTTCTTCTAAATCCCAACCGAACTTCTTCCAACCTCCAAACTG GCTGTAACCCCAAATCCAAGCCATTAACTACACCTGACAGTAGCAATTGTCTGATTAATCAC TGGCCCCTTGAAGACAGCAGAATGTCCCTTTGCAATGAGGAGGAGATCTGGGCTGGGCGGG CCAGCTGGGGAAGCATTTGACTATCTGGAACTTGTGTGTGCCTCCTCAGGTATGGCAGTGACT CACCTGGTTTTAATAAAACAACCTGCAACATCTCATGGTCTTTGAATAAAGCCTGAGTAGGA AGTCTAGA 3UTR- α-actinACACACTCCACCTCCAGCACGCGACTTCTCA  7 003 GGACGACGAATCTTCTCAATGGGGGGGCGGCTGAGCTCCAGCCACCCCGCAGTCACTTTCTTT GTAACAACTTCCGTTGCTGCCATCGTAAACTGACACAGTGTTTATAACGTGTACATACATTA ACTTATTACCTCATTTTGTTATTTTTCGAAACAAAGCCCTGTGGAAGAAAATGGAAAACTTG AAGAAGCATTAAAGTCATTCTGTTAAGCTGCGTAAATGGTCTTTGAATAAAGCCTGAGTAGG AAGTCTAGA 3UTR- AlbuminCATCACATTTAAAAGCATCTCAGCCTACCAT  8 004 GAGAATAAGAGAAAGAAAATGAAGATCAAAAGCTTATTCATCTGTTTTTCTTTTTCGTTGGTG TAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGC TTCAATTAATAAAAAATGGAAAGAATCTAATAGAGTGGTACAGCACTGTTATTTTTCAAAGA TGTGTTGCTATCCTGAAAATTCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCTTATTCCACT TCGGTAGAGGATTTCTAGTTTCTTGTGGGCTAATTAAATAAATCATTAATACTCTTCTAATGGT CTTTGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-α-globin GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCAT  9 005GCCCTTCTTCTCTCCCTTGCACCTGTACCTCT TGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 3UTR- G-CSF GCCAAGCCCTCCCCATCCCATGTATTTATCTC 10006 TATTTAATATTTATGTCTATTTAAGCCTCATA TTTAAAGACAGGGAAGAGCAGAACGGAGCCCCAGGCCTCTGTGTCCTTCCCTGCATTTCTGA GTTTCATTCTCCTGCCTGTAGCAGTGAGAAAAAGCTCCTGTCCTCCCATCCCCTGGACTGGG AGGTAGATAGGTAAATACCAAGTATTTATTACTATGACTGCTCCCCAGCCCTGGCTCTGCAAT GGGCACTGGGATGAGCCGCTGTGAGCCCCTGGTCCTGAGGGTCCCCACCTGGGACCCTTGAG AGTATCAGGTCTCCCACGTGGGAGACAAGAAATCCCTGTTTAATATTTAAACAGCAGTGTTCC CCATCTGGGTCCTTGCACCCCTCACTCTGGCCTCAGCCGACTGCACAGCGGCCCCTGCATCCC CTTGGCTGTGAGGCCCCTGGACAAGCAGAGGTGGCCAGAGCTGGGAGGCATGGCCCTGGGGT CCCACGAATTTGCTGGGGAATCTCGTTTTTCTTCTTAAGACTTTTGGGACATGGTTTGACTCCC GAACATCACCGACGCGTCTCCTGTTTTTCTGGGTGGCCTCGGGACACCTGCCCTGCCCCCACG AGGGTCAGGACTGTGACTCTTTTTAGGGCCAGGCAGGTGCCTGGACATTTGCCTTGCTGGAC GGGGACTGGGGATGTGGGAGGGAGCAGACAGGAGGAATCATGTCAGGCCTGTGTGTGAAAG GAAGCTCCACTGTCACCCTCCACCTCTTCACCCCCCACTCACCAGTGTCCCCTCCACTGTCACA TTGTAACTGAACTTCAGGATAATAAAGTGTTTGCCTCCATGGTCTTTGAATAAAGCCTGAGT AGGAAGGCGGCCGCTCGAGCATGCATCTAGA 3UTR-COl1a2 ACTCAATCTAAATTAAAAAAGAAAGAAATTT 11 007 collagen,GAAAAAACTTTCTCTTTGCCATTTCTTCTTCT type TCTTTTTTAACTGAAAGCTGAATCCTTCCATTI alpha 2 TCTTCTGCACATCTACTTGCTTAAATTGTGGGCAAAAGAGAAAAAGAAGGATTGATCAGAGC ATTGTGCAATACAGTTTCATTAACTCCTTCCCCCGCTCCCCCAAAAATTTGAATTTTTTTTTCA ACACTCTTACACCTGTTATGGAAAATGTCAACCTTTGTAAGAAAACCAAAATAAAAATTGAA AAATAAAAACCATAAACATTTGCACCACTTGTGGCTTTTGAATATCTTCCACAGAGGGAAGT TTAAAACCCAAACTTCCAAAGGTTTAAACTACCTCAAAACACTTTCCCATGAGTGTGATCCA CATTGTTAGGTGCTGACCTAGACAGAGATGAACTGAGGTCCTTGTTTTGTTTTGTTCATAATA CAAAGGTGCTAATTAATAGTATTTCAGATACTTGAAGAATGTTGATGGTGCTAGAAGAATTT GAGAAGAAATACTCCTGTATTGAGTTGTATCGTGTGGTGTATTTTTTAAAAAATTTGATTTAG CATTCATATTTTCCATCTTATTCCCAATTAAAAGTATGCAGATTATTTGCCCAAATCTTCTTCA GATTCAGCATTTGTTCTTTGCCAGTCTCATTTTCATCTTCTTCCATGGTTCCACAGAAGCTTTG TTTCTTGGGCAAGCAGAAAAATTAAATTGTACCTATTTTGTATATGTGAGATGTTTAAATAAA TTGTGAAAAAAATGAAATAAAGCATGTTTGGTTTTCCAAAAGAACATAT 3UTR- Col6a2; CGCCGCCGCCCGGGCCCCGCAGTCGAGGGTC 12 008collagen, GTGAGCCCACCCCGTCCATGGTGCTAAGCGG typeGCCCGGGTCCCACACGGCCAGCACCGCTGCT VI alpha 2CACTCGGACGACGCCCTGGGCCTGCACCTCT CCAGCTCCTCCCACGGGGTCCCCGTAGCCCCGGCCCCCGCCCAGCCCCAGGTCTCCCCAGGC CCTCCGCAGGCTGCCCGGCCTCCCTCCCCCTGCAGCCATCCCAAGGCTCCTGACCTACCTGGC CCCTGAGCTCTGGAGCAAGCCCTGACCCAATAAAGGCTTTGAACCCAT 3UTR- RPN1; GGGGCTAGAGCCCTCTCCGCACAGCGTGGAG 13 009ribophorin ACGGGGCAAGGAGGGGGGTTATTAGGATTG IGTGGTTTTGTTTTGCTTTGTTTAAAGCCGTGG GAAAATGGCACAACTTTACCTCTGTGGGAGATGCAACACTGAGAGCCAAGGGGTGGGAGTT GGGATAATTTTTATATAAAAGAAGTTTTTCCACTTTGAATTGCTAAAAGTGGCATTTTTCCTA TGTGCAGTCACTCCTCTCATTTCTAAAATAGGGACGTGGCCAGGCACGGTGGCTCATGCCTGT AATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGCTCACGAGGTCAGGAGATCGAGACTATCC TGGCTAACACGGTAAAACCCTGTCTCTACTAAAAGTACAAAAAATTAGCTGGGCGTGGTGGT GGGCACCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAAAGGCATGAATCCAAGAG GCAGAGCTTGCAGTGAGCTGAGATCACGCCATTGCACTCCAGCCTGGGCAACAGTGTTAAGA CTCTGTCTCAAATATAAATAAATAAATAAATAAATAAATAAATAAATAAAAATAAAGCGAG ATGTTGCCCTCAAA 3UTR- LRP1;lowGGCCCTGCCCCGTCGGACTGCCCCCAGAAAG 14 010 densityCCTCCTGCCCCCTGCCAGTGAAGTCCTTCAGT lipoproteinGAGCCCCTCCCCAGCCAGCCCTTCCCTGGCC receptor-CCGCCGGATGTATAAATGTAAAAATGAAGGA related ATTACATTTTATATGTGAGCGAGCAAGCCGGprotein 1 CAAGCGAGCACAGTATTATTTCTCCATCCCCTCCCTGCCTGCTCCTTGGCACCCCCATGCTGCC TTCAGGGAGACAGGCAGGGAGGGCTTGGGGCTGCACCTCCTACCCTCCCACCAGAACGCAC CCCACTGGGAGAGCTGGTGGTGCAGCCTTC CCCTCCCTGTATAAGACACTTTGCCAAGGCTCT CCCCTCTCGCCCCATCCCTGCTTGCCCGCTCCCACAGCTTCCTGAGGGCTAATTCTGGGAAGG GAGAGTTCTTTGCTGCCCCTGTCTGGAAGACGTGGCTCTGGGTGAGGTAGGCGGGAAAGGA TGGAGTGTTTTAGTTCTTGGGGGAGGCCACCCCAAACCCCAGCCCCAACTCCAGGGGCACCT ATGAGATGGCCATGCTCAACCCCCCTCCCAGACAGGCCCTCCCTGTCTCCAGGGCCCCCACC GAGGTTCCCAGGGCTGGAGACTTCCTCTGGTAAACATTCCTCCAGCCTCCCCTCCCCTGGGG ACGCCAAGGAGGTGGGCCACACCCAGGAAGGGAAAGCGGGCAGCCCCGTTTTGGGGACGTG AACGTTTTAATAATTTTTGCTGAATTCCTTTACAACTAAATAACACAGATATTGTTATAAATA AAATTGT 3UTR- Nnt1;ATATTAAGGATCAAGCTGTTAGCTAATAATG 15 011 cardio-CCACCTCTGCAGTTTTGGGAACAGGCAAATA trophin- AAGTATCAGTATACATGGTGATGTACATCTGlike  TAGCAAAGCTCTTGGAGAAAATGAAGACTGA cytokineAGAAAGCAAAGCAAAAACTGTATAGAGAGA factor 1 TTTTTCAAAAGCAGTAATCCCTCAATTTTAAAAAAGGATTGAAAATTCTAAATGTCTTTCTGT GCATATTTTTTGTGTTAGGAATCAAAAGTATTTTATAAAAGGAGAAAGAACAGCCTCATTTTA GATGTAGTCCTGTTGGATTTTTTATGCCTCCTCAGTAACCAGAAATGTTTTAAAAAACTAAGT GTTTAGGATTTCAAGACAACATTATACATGGCTCTGAAATATCTGACACAATGTAAACATTG CAGGCACCTGCATTTTATGTTTTTTTTTTCAACAAATGTGACTAATTTGAAACTTTTATGAAC TTCTGAGCTGTCCCCTTGCAATTCAACCGCAGTTTGAATTAATCATATCAAATCAGTTTTAATT TTTTAAATTGTACTTCAGAGTCTATATTTCAA GGGCACATTTTCTCACTACTATTTTAATACAT TAAAGGACTAAATAATCTTTCAGAGATGCTGGAAACAAATCATTTGCTTTATATGTTTCATTA GAATACCAATGAAACATACAACTTGAAAATTAGTAATAGTATTTTTGAAGATCCCATTTCTAA TTGGAGATCTCTTTAATTTCGATCAACTTATAATGTGTAGTACTATATTAAGTGCACTTGAGT GGAATTCAACATTTGACTAATAAAATGAGTTCATCATGTTGGCAAGTGATGTGGCAATTATC TCTGGTGACAAAAGAGTAAAATCAAATATTTCTGCCTGTTACAAATATCAAGGAAGACCTGC TACTATGAAATAGATGACATTAATCTGTCTTCACTGTTTATAATACGGATGGATTTTTTTTCAA ATCAGTGTGTGTTTTGAGGTCTTATGTAATTGATGACATTTGAGAGAAATGGTGGCTTTTTTT AGCTACCTCTTTGTTCATTTAAGCACCAGTAAAGATCATGTCTTTTTATAGAAGTGTAGATTTT CTTTGTGACTTTGCTATCGTGCCTAAAGCTCTAAATATAGGTGAATGTGTGATGAATACTCAG ATTATTTGTCTCTCTATATAATTAGTTTGGTACTAAGTTTCTCAAAAAATTATTAACACATGA AAGACAATCTCTAAACCAGAAAAAGAAGTAGTACAAATTTTGTTACTGTAATGCTCGCGTTT AGTGAGTTTAAAACACACAGTATCTTTTGGTTTTATAATCAGTTTCTATTTTGCTGTGCCTGA GATTAAGATCTGTGTATGTGTGTGTGTGTGTGTGTGCGTTTGTGTGTTAAAGCAGAAAAGACT TTTTTAAAAGTTTTAAGTGATAAATGCAATTTGTTAATTGATCTTAGATCACTAGTAAACTCA GGGCTGAATTATACCATGTATATTCTATTAGAAGAAAGTAAACACCATCTTTATTCCTGCCC TTTTTCTTCTCTCAAAGTAGTTGTAGTTATATCTAGAAAGAAGCAATTTTGATTTCTTGAAAA GGTAGTTCCTGCACTCAGTTTAAACTAAAAATAATCATACTTGGATTTTATTTATTTTTGTCA TAGTAAAAATTTTAATTTATATATATTTTTATTTAGTATTATCTTATTCTTTGCTATTTGCCAA TCCTTTGTCATCAATTGTGTTAAATGAATTGAAAATTCATGCCCTGTTCATTTTATTTTACTTT ATTGGTTAGGATATTTAAAGGATTTTTGTATATATAATTTCTTAAATTAATATTCCAAAAGGTT AGTGGACTTAGATTATAAATTATGGCAAAAATCTAAAAACAACAAAAATGATTTTTATACAT TCTATTTCATTATTCCTCTTTTTCCAATAAGTCATACAATTGGTAGATATGACTTATTTTATTTT TGTATTATTCACTATATCTTTATGATATTTAAGTATAAATAATTAAAAAAATTTATTGTACCT TATAGTCTGTCACCAAAAAAAAAAAATTATCTGTAGGTAGTGAAATGCTAATGTTGATTTGT CTTTAAGGGCTTGTTAACTATCCTTTATTTTCTCATTTGTCTTAAATTAGGAGTTTGTGTTTAA ATTACTCATCTAAGCAAAAAATGTATATAAATCCCATTACTGGGTATATACCCAAAGGATTA TAAATCATGCTGCTATAAAGACACATGCACACGTATGTTTATTGCAGCACTATTCACAATAGC AAAGACTTGGAACCAACCCAAATGTCCATCAATGATAGACTTGATTAAGAAAATGTGCACAT ATACACCATGGAATACTATGCAGCCATAAAAAAGGATGAGTTCATGTCCTTTGTAGGGACAT GGATAAAGCTGGAAACCATCATTCTGAGCAAACTATTGCAAGGACAGAAAACCAAACACTGC ATGTTCTCACTCATAGGTGGGAATTGAACAATGAGAACACTTGGACACAAGGTGGGGAACA CCACACACCAGGGCCTGTCATGGGGTGGGGGGAGTGGGGAGGGATAGCATTAGGAGATATA CCTAATGTAAATGATGAGTTAATGGGTGCAGCACACCAACATGGCACATGTATACATATGTA GCAAACCTGCACGTTGTGCACATGTACCCTAGAACTTAAAGTATAATTAAAAAAAAAAAGA AAACAGAAGCTATTTATAAAGAAGTTATTTGCTGAAATAAATGTGATCTTTCCCATTAAAAA AATAAAGAAATTTTGGGGTAAAAAAACACAATATATTGTATTCTTGAAAAATTCTAAGAGA GTGGATGTGAAGTGTTCTCACCACAAAAGTGATAACTAATTGAGGTAATGCACATATTAATT AGAAAGATTTTGTCATTCCACAATGTATATATACTTAAAAATATGTTATACACAATAAATAC ATACATTAAAAAATAAGTAAATGTA 3UTR- Col6a1;CCCACCCTGCACGCCGGCACCAAACCCTGTC 16 012 collagen,CTCCCACCCCTCCCCACTCATCACTAAACAG type AGTAAAATGTGATGCGAATTTTCCCGACCAAVI alpha 1, CCTGATTCGCTAGATTTTTTTTAAGGAAAAGCTTGGAAAGCCAGGACACAACGCTGCTGCCTG CTTTGTGCAGGGTCCTCCGGGGCTCAGCCCTGAGTTGGCATCACCTGCGCAGGGCCCTCTGG GGCTCAGCCCTGAGCTAGTGTCACCTGCACAGGGCCCTCTGAGGCTCAGCCCTGAGCTGGCG TCACCTGTGCAGGGCCCTCTGGGGCTCAGCCCTGAGCTGGCCTCACCTGGGTTCCCCACCCC GGGCTCTCCTGCCCTGCCCTCCTGCCCGCCCTCCCTCCTGCCTGCGCAGCTCCTTCCCTAGGCA CCTCTGTGCTGCATCCCACCAGCCTGAGCAAGACGCCCTCTCGGGGCCTGTGCCGCACTAGC CTCCCTCTCCTCTGTCCCCATAGCTGGTTTTTCCCACCAATCCTCACCTAACAGTTACTTTACA ATTAAACTCAAAGCAAGCTCTTCTCCTCAGCTTGGGGCAGCCATTGGCCTCTGTCTCGTTTTG GGAAACCAAGGTCAGGAGGCCGTTGCAGACATAAATCTCGGCGACTCGGCCCCGTCTCCTG AGGGTCCTGCTGGTGACCGGCCTGGACCTTGGCCCTACAGCCCTGGAGGCCGCTGCTGACCA GCACTGACCCCGACCTCAGAGAGTACTCGCAGGGGCGCTGGCTGCACTCAAGACCCTCGAGA TTAACGGTGCTAACCCCGTCTGCTCCTCCCTCCCGCAGAGACTGGGGCCTGGACTGGACATGA GAGCCCCTTGGTGCCACAGAGGGCTGTGTCTTACTAGAAACAACGCAAACCTCTCCTTCCTC AGAATAGTGATGTGTTCGACGTTTTATCAAAGGCCCCCTTTCTATGTTCATGTTAGTTTTGCT CCTTCTGTGTTTTTTTCTGAACCATATCCATGTTGCTGACTTTTCCAAATAAAGGTTTTCACTC CTCTC 3UTR- Calr;AGAGGCCTGCCTCCAGGGCTGGACTGAGGCC 17 013 calreticulinTGAGCGCTCCTGCCGCAGAGCTGGCCGCGCC AAATAATGTCTCTGTGAGACTCGAGAACTTTCATTTTTTTCCAGGCTGGTTCGGATTTGGGGTT GGATTTTGGTTTTGTTCCCCTCCTCCACTCTCCCCCACCCCCTCCCCGCCCTTTTTTTTTTTTTT TTTTAAACTGGTATTTTATCTTTGATTCTCCTTCAGCCCTCACCCCTGGTTCTCATCTTTCTTGA TCAACATCTTTTCTTGCCTCTGTCCCCTTCTCTCATCTCTTAGCTCCCCTCCAACCTGGGGGGC AGTGGTGTGGAGAAGCCACAGGCCTGAGATTTCATCTGCTCTCCTTCCTGGAGCCCAGAGGA GGGCAGCAGAAGGGGGTGGTGTCTCCAACCCCCCAGCACTGAGGAAGAACGGGGCTCTTCTC ATTTCACCCCTCCCTTTCTCCCCTGCCCCCAGGACTGGGCCACTTCTGGGTGGGGCAGTGGGT CCCAGATTGGCTCACACTGAGAATGTAAGAACTACAAACAAAATTTCTATTAAATTAAATTTT GTGTCTCC 3UTR- Col1a1;CTCCCTCCATCCCAACCTGGCTCCCTCCCACC 18 014 collagen,CAACCAACTTTCCCCCCAACCCGGAAACAGA type CAAGCAACCCAAACTGAACCCCCTCAAAAGCI, alpha 1 CAAAAAATGGGAGACAATTTCACATGGACTTTGGAAAATATTTTTTTCCTTTGCATTCATCTC TCAAACTTAGTTTTTATCTTTGACCAACCGAACATGACCAAAAACCAAAAGTGCATTCAACCT TACCAAAAAAAAAAAAAAAAAAAGAATAAATAAATAACTTTTTAAAAAAGGAAGCTTGGTC CACTTGCTTGAAGACCCATGCGGGGGTAAGTCCCTTTCTGCCCGTTGGGCTTATGAAACCCCA ATGCTGCCCTTTCTGCTCCTTTCTCCACACCCCCCTTGGGGCCTCCCCTCCACTCCTTCCCAAA TCTGTCTCCCCAGAAGACACAGGAAACAATGTATTGTCTGCCCAGCAATCAAAGGCAATGCT CAAACACCCAAGTGGCCCCCACCCTCAGCCCGCTCCTGCCCGCCCAGCACCCCCAGGCCCTG GGGGACCTGGGGTTCTCAGACTGCCAAAGAAGCCTTGCCATCTGGCGCTCCCATGGCTCTTGC AACATCTCCCCTTCGTTTTTGAGGGGGTCATGCCGGGGGAGCCACCAGCCCCTCACTGGGTTC GGAGGAGAGTCAGGAAGGGCCACGACAAAGCAGAAACATCGGATTTGGGGAACGCGTGTCA ATCCCTTGTGCCGCAGGGCTGGGCGGGAGAGACTGTTCTGTTCCTTGTGTAACTGTGTTGCTG AAAGACTACCTCGTTCTTGTCTTGATGTGTCACCGGGGCAACTGCCTGGGGGCGGGGATGGG GGCAGGGTGGAAGCGGCTCCCCATTTTATACCAAAGGTGCTACATCTATGTGATGGGTGGGG TGGGGAGGGAATCACTGGTGCTATAGAAATTGAGATGCCCCCCCAGGCCAGCAAATGTTCCT TTTTGTTCAAAGTCTATTTTTATTCCTTGATATTTTTCTTTTTTTTTTTTTTTTTTTGTGGATGGG GACTTGTGAATTTTTCTAAAGGTGCTATTTAACATGGGAGGAGAGCGTGTGCGGCTCCAGCCC AGCCCGCTGCTCACTTTCCACCCTCTCTCCACCTGCCTCTGGCTTCTCAGGCCTCTGCTCTCCG ACCTCTCTCCTCTGAAACCCTCCTCCACAGCTGCAGCCCATCCTCCCGGCTCCCTCCTAGTCTG TCCTGCGTCCTCTGTCCCCGGGTTTCAGAGACAACTTCCCAAAGCACAAAGCAGTTTTTCCCC CTAGGGGTGGGAGGAAGCAAAAGACTCTGTACCTATTTTGTATGTGTATAATAATTTGAGAT GTTTTTAATTATTTTGATTGCTGGAATAAAGCATGTGGAAATGACCCAAACATAATCCGCAGT GGCCTCCTAATTTCCTTCTTTGGAGTTGGGGGAGGGGTAGACATGGGGAAGGGGCTTTGGGG TGATGGGCTTGCCTTCCATTCCTGCCCTTTCCCTCCCCACTATTCTCTTCTAGATCCCTCCATA ACCCCACTCCCCTTTCTCTCACCCTTCTTATACCGCAAACCTTTCTACTTCCTCTTTCATTTTCT ATTCTTGCAATTTCCTTGCACCTTTTCCAAATCCTCTTCTCCCCTGCAATACCATACAGGCAAT CCACGTGCACAACACACACACACACTCTTCACATCTGGGGTTGTCCAAACCTCATACCCACT CCCCTTCAAGCCCATCCACTCTCCACCCCCTGGATGCCCTGCACTTGGTGGCGGTGGGATGCT CATGGATACTGGGAGGGTGAGGGGAGTGGAACCCGTGAGGAGGACCTGGGGGCCTCTCCTT GAACTGACATGAAGGGTCATCTGGCCTCTGCTCCCTTCTCACCCACGCTGACCTCCTGCCGAA GGAGCAACGCAACAGGAGAGGGGTCTGCTGAGCCTGGCGAGGGTCTGGGAGGGACCAGGA GGAAGGCGTGCTCCCTGCTCGCTGTCCTGGCCCTGGGGGAGTGAGGGAGACAGACACCTGG GAGAGCTGTGGGGAAGGCACTCGCACCGTGCTCTTGGGAAGGAAGGAGACCTGGCCCTGCTC ACCACGGACTGGGTGCCTCGACCTCCTGAATCCCCAGAACACAACCCCCCTGGGCTGGGGTG GTCTGGGGAACCATCGTGCCCCCGCCTCCCGCCTACTCCTTTTTAAGCTT 3UTR- Plod1; TTGGCCAGGCCTGACCCTCTTGGACCTTTCTT 19 015procollagen- CTTTGCCGACAACCACTGCCCAGCAGCCTCT lysine, 2-GGGACCTCGGGGTCCCAGGGAACCCAGTCCA oxoglutarate GCCTCCTGGCTGTTGACTTCCCATTGCTCTTG 5- GAGCCACCAATCAAAGAGATTCAAAGAGATTdioxygenase  CCTGCAGGCCAGAGGCGGAACACACCTTTAT 1GGCTGGGGCTCTCCGTGGTGTTCTGGACCCA GCCCCTGGAGACACCATTCACTTTTACTGCTTTGTAGTGACTCGTGCTCTCCAACCTGTCTTCC TGAAAAACCAAGGCCCCCTTCCCCCACCTCTTCCATGGGGTGAGACTTGAGCAGAACAGGG GCTTCCCCAAGTTGCCCAGAAAGACTGTCTGGGTGAGAAGCCATGGCCAGAGCTTCTCCCAG GCACAGGTGTTGCACCAGGGACTTCTGCTTCAAGTTTTGGGGTAAAGACACCTGGATCAGAC TCCAAGGGCTGCCCTGAGTCTGGGACTTCTGCCTCCATGGCTGGTCATGAGAGCAAACCGTA GTCCCCTGGAGACAGCGACTCCAGAGAACCTCTTGGGAGACAGAAGAGGCATCTGTGCACAG CTCGATCTTCTACTTGCCTGTGGGGAGGGGAGTGACAGGTCCACACACCACACTGGGTCACC CTGTCCTGGATGCCTCTGAAGAGAGGGACAGACCGTCAGAAACTGGAGAGTTTCTATTAAAG GTCATTTAAACCA 3UTR- Nucb1;TCCTCCGGGACCCCAGCCCTCAGGATTCCTG 20 016 nucleobindinATGCTCCAAGGCGACTGATGGGCGCTGGATG 1 AAGTGGCACAGTCAGCTTCCCTGGGGGCTGGTGTCATGTTGGGCTCCTGGGGCGGGGGCACG GCCTGGCATTTCACGCATTGCTGCCACCCCAGGTCCACCTGTCTCCACTTTCACAGCCTCCAA GTCTGTGGCTCTTCCCTTCTGTCCTCCGAGGGGCTTGCCTTCTCTCGTGTCCAGTGAGGTGCTC AGTGATCGGCTTAACTTAGAGAAGCCCGCCCCCTCCCCTTCTCCGTCTGTCCCAAGAGGGTCT GCTCTGAGCCTGCGTTCCTAGGTGGCTCGGCCTCAGCTGCCTGGGTTGTGGCCGCCCTAGCA TCCTGTATGCCCACAGCTACTGGAATCCCCGCTGCTGCTCCGGGCCAAGCTTCTGGTTGATTA  ATGAGGGCATGGGGTGGTCCCTCAAGACCTTCCCCTACCTTTTGTGGAACCAGTGATGCCTCA AAGACAGTGTCCCCTCCACAGCTGGGTGCCAGGGGCAGGGGATCCTCAGTATAGCCGGTGAA CCCTGATACCAGGAGCCTGGGCCTCCCTGAACCCCTGGCTTCCAGCCATCTCATCGCCAGCCT CCTCCTGGACCTCTTGGCCCCCAGCCCCTTCCCCACACAGCCCCAGAAGGGTCCCAGAGCTGA CCCCACTCCAGGACCTAGGCCCAGCCCCTCAGCCTCATCTGGAGCCCCTGAAGACCAGTCCC ACCCACCTTTCTGGCCTCATCTGACACTGCTCCGCATCCTGCTGTGTGTCCTGTTCCATGTTCC GGTTCCATCCAAATACACTTTCTGGAACAAA 3UTR-α-globin GCTGGAGCCTCGGTGGCCATGCTTCTTGCCC 21 017CTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTC CTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC

It should be understood that those listed in the previous tables areexamples and that any UTR from any gene may be incorporated into therespective first or second flanking region of the primary construct.Furthermore, multiple wild-type UTRs of any known gene may be utilized.It is also within the scope of the present invention to provideartificial UTRs which are not variants of wild type genes. These UTRs orportions thereof may be placed in the same orientation as in thetranscript from which they were selected or may be altered inorientation or location. Hence a 5′ or 3′ UTR may be inverted,shortened, lengthened, made chimeric with one or more other 5′ UTRs or3′ UTRs. As used herein, the term “altered” as it relates to a UTRsequence, means that the UTR has been changed in some way in relation toa reference sequence. For example, a 3′ or 5′ UTR may be alteredrelative to a wild type or native UTR by the change in orientation orlocation as taught above or may be altered by the inclusion ofadditional nucleotides, deletion of nucleotides, swapping ortransposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In one embodiment, a double, triple or quadruple UTR such as a 5′ or 3′UTR may be used. As used herein, a “double” UTR is one in which twocopies of the same UTR are encoded either in series or substantially inseries. For example, a double beta-globin 3′ UTR may be used asdescribed in US Patent publication 20100129877, the contents of whichare incorporated herein by reference in its entirety.

It is also within the scope of the present invention to have patternedUTRs. As used herein “patterned UTRs” are those UTRs which reflect arepeating or alternating pattern, such as ABABAB or AABBAABBAABB orABCABCABC or variants thereof repeated once, twice, or more than 3times. In these patterns, each letter, A, B, or C represent a differentUTR at the nucleotide level.

In one embodiment, flanking regions are selected from a family oftranscripts whose proteins share a common function, structure, featureof property. For example, oncology-related polypeptides of interest maybelong to a family of proteins which are expressed in a particular cell,tissue or at some time during development. The UTRs from any of thesegenes may be swapped for any other UTR of the same or different familyof proteins to create a new chimeric primary transcript. As used herein,a “family of proteins” is used in the broadest sense to refer to a groupof two or more oncology-related polypeptides of interest which share atleast one function, structure, feature, localization, origin, orexpression pattern.

After optimization (if desired), the oncology-related primary constructcomponents are reconstituted and transformed into a vector such as, butnot limited to, plasmids, viruses, cosmids, and artificial chromosomes.For example, the optimized construct may be reconstituted andtransformed into chemically competent E. coli, yeast, neurospora, maize,drosophila, etc. where high copy plasmid-like or chromosome structuresoccur by methods described herein.

The untranslated region may also include translation enhancer elements(TEE). As a non-limiting example, the TEE may include those described inUS Application No. 20090226470, herein incorporated by reference in itsentirety, and those known in the art.

Stop Codons

In one embodiment, the oncology-related primary constructs of thepresent invention may include at least two stop codons before the 3′untranslated region (UTR). The stop codon may be selected from TGA, TAAand TAG. In one embodiment, the oncology-related primary constructs ofthe present invention include the stop codon TGA and one additional stopcodon. In a further embodiment the addition stop codon may be TAA. Inanother embodiment, the primary constructs of the present inventioninclude three stop codons.

Vector Amplification

The vector containing the oncology-related primary construct is thenamplified and the plasmid isolated and purified using methods known inthe art such as, but not limited to, a maxi prep using the InvitrogenPURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.).

Plasmid Linearization

The plasmid may then be linearized using methods known in the art suchas, but not limited to, the use of restriction enzymes and buffers. Thelinearization reaction may be purified using methods including, forexample Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.), andHPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen'sstandard PURELINK™ PCR Kit (Carlsbad, Calif.). The purification methodmay be modified depending on the size of the linearization reactionwhich was conducted. The linearized plasmid is then used to generatecDNA for in vitro transcription (IVT) reactions.

cDNA Template Synthesis

A cDNA template may be synthesized by having a linearized plasmidundergo polymerase chain reaction (PCR). Table 4 is a listing of primersand probes that may be useful in the PCR reactions of the presentinvention. It should be understood that the listing is not exhaustiveand that primer-probe design for any amplification is within the skillof those in the art. Probes may also contain chemically modified basesto increase base-pairing fidelity to the target molecule andbase-pairing strength. Such modifications may include 5-methyl-Cytidine,2,6-di-amino-purine, 2′-fluoro, phosphoro-thioate, or locked nucleicacids.

TABLE 4 Primers and Probes Primer/ SEQ Probe Hybridization ID IdentifierSequence (5′-3′) target NO. UFP TTGGACCCTCGTACAGAAGCTAA cDNA Template 22TACG URP T_(x160)CTTCCTACTCAGGCTTTATTC cDNA Template 23 AAAGACCA GBA1CCTTGACCTTCTGGAACTTC Acid 24 glucocerebrosidase GBA2CCAAGCACTGAAACGGATAT Acid 25 glucocerebrosidase LUC1GATGAAAAGTGCTCCAAGGA Luciferase 26 LUC2 AACCGTGATGAAAAGGTACC Luciferase27 LUC3 TCATGCAGATTGGAAAGGTC Luciferase 28 GCSF1 CTTCTTGGACTGTCCAGAGGG-CSF 29 GCSF2 GCAGTCCCTGATACAAGAAC G-CSF 30 GCSF3 GATTGAAGGTGGCTCGCTACG-CSF 31 *UFPis universal forward primer; URP is universal reverseprimer.

In one embodiment, the cDNA may be submitted for sequencing analysisbefore undergoing transcription.

mRNA Production

The process of mRNA or mmRNA production may include, but is not limitedto, in vitro transcription, cDNA template removal and RNA clean-up, andmRNA capping and/or tailing reactions.

In Vitro Transcription

The cDNA produced in the previous step may be transcribed using an invitro transcription (IVT) system. The system typically comprises atranscription buffer, nucleotide triphosphates (NTPs), an RNaseinhibitor and a polymerase. The NTPs may be manufactured in house, maybe selected from a supplier, or may be synthesized as described herein.The NTPs may be selected from, but are not limited to, those describedherein including natural and unnatural (modified) NTPs. The polymerasemay be selected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to be incorporated into modified nucleic acids.

RNA Polymerases

Any number of RNA polymerases or variants may be used in the design ofthe oncology-related primary constructs of the present invention.

RNA polymerases may be modified by inserting or deleting amino acids ofthe RNA polymerase sequence. As a non-limiting example, the RNApolymerase may be modified to exhibit an increased ability toincorporate a 2′-modified nucleotide triphosphate compared to anunmodified RNA polymerase (see International Publication WO2008078180and U.S. Pat. No. 8,101,385; herein incorporated by reference in theirentireties).

Variants may be obtained by evolving an RNA polymerase, optimizing theRNA polymerase amino acid and/or nucleic acid sequence and/or by usingother methods known in the art. As a non-limiting example, T7 RNApolymerase variants may be evolved using the continuous directedevolution system set out by Esvelt et al. (Nature (2011)472(7344):499-503; herein incorporated by reference in its entirety)where clones of T7 RNA polymerase may encode at least one mutation suchas, but not limited to, lysine at position 93 substituted for threonine(K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T,N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A,Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P,A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A,H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E,N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limitingexample, T7 RNA polymerase variants may encode at least mutation asdescribed in U.S. Pub. Nos. 20100120024 and 20070117112; hereinincorporated by reference in their entireties. Variants of RNApolymerase may also include, but are not limited to, substitutionalvariants, conservative amino acid substitution, insertional variants,deletional variants and/or covalent derivatives.

In one embodiment, the oncology-related primary construct may bedesigned to be recognized by the wild type or variant RNA polymerases.In doing so, the oncology-related primary construct may be modified tocontain sites or regions of sequence changes from the wild type orparent primary construct.

In one embodiment, the oncology-related primary construct may bedesigned to include at least one substitution and/or insertion upstreamof an RNA polymerase binding or recognition site, downstream of the RNApolymerase binding or recognition site, upstream of the TATA boxsequence, downstream of the TATA box sequence of the oncology-relatedprimary construct but upstream of the coding region of theoncology-related primary construct, within the 5′UTR, before the 5′UTRand/or after the 5′UTR.

In one embodiment, the 5′UTR of the oncology-related primary constructmay be replaced by the insertion of at least one region and/or string ofnucleotides of the same base. The region and/or string of nucleotidesmay include, but is not limited to, at least 3, at least 4, at least 5,at least 6, at least 7 or at least 8 nucleotides and the nucleotides maybe natural and/or unnatural. As a non-limiting example, the group ofnucleotides may include 5-8 adenine, cytosine, thymine, a string of anyof the other nucleotides disclosed herein and/or combinations thereof.

In one embodiment, the 5′UTR of the oncology-related primary constructmay be replaced by the insertion of at least two regions and/or stringsof nucleotides of two different bases such as, but not limited to,adenine, cytosine, thymine, any of the other nucleotides disclosedherein and/or combinations thereof. For example, the 5′UTR may bereplaced by inserting 5-8 adenine bases followed by the insertion of 5-8cytosine bases. In another example, the 5′UTR may be replaced byinserting 5-8 cytosine bases followed by the insertion of 5-8 adeninebases.

In one embodiment, the oncology-related primary construct may include atleast one substitution and/or insertion downstream of the transcriptionstart site which may be recognized by an RNA polymerase. As anon-limiting example, at least one substitution and/or insertion mayoccur downstream the transcription start site by substituting at leastone nucleic acid in the region just downstream of the transcriptionstart site (such as, but not limited to, +1 to +6). Changes to region ofnucleotides just downstream of the transcription start site may affectinitiation rates, increase apparent nucleotide triphosphate (NTP)reaction constant values, and increase the dissociation of shorttranscripts from the transcription complex curing initial transcription(Brieba et al, Biochemistry (2002) 41: 5144-5149; herein incorporated byreference in its entirety). The modification, substitution and/orinsertion of at least one nucleic acid may cause a silent mutation ofthe nucleic acid sequence or may cause a mutation in the amino acidsequence.

In one embodiment, the oncology-related primary construct may includethe substitution of at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12 or at least 13 guanine bases downstream of thetranscription start site.

In one embodiment, the oncology-related primary construct may includethe substitution of at least 1, at least 2, at least 3, at least 4, atleast 5 or at least 6 guanine bases in the region just downstream of thetranscription start site. As a non-limiting example, if the nucleotidesin the region are GGGAGA the guanine bases may be substituted by atleast 1, at least 2, at least 3 or at least 4 adenine nucleotides. Inanother non-limiting example, if the nucleotides in the region areGGGAGA the guanine bases may be substituted by at least 1, at least 2,at least 3 or at least 4 cytosine bases. In another non-limitingexample, if the nucleotides in the region are GGGAGA the guanine basesmay be substituted by at least 1, at least 2, at least 3 or at least 4thymine, and/or any of the nucleotides described herein.

In one embodiment, the oncology-related primary construct may include atleast one substitution and/or insertion upstream of the start codon. Forthe purpose of clarity, one of skill in the art would appreciate thatthe start codon is the first codon of the protein coding region whereasthe transcription start site is the site where transcription begins. Theoncology-related primary construct may include, but is not limited to,at least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7 or at least 8 substitutions and/or insertions of nucleotidebases. The nucleotide bases may be inserted or substituted at 1, atleast 1, at least 2, at least 3, at least 4 or at least 5 locationsupstream of the start codon. The nucleotides inserted and/or substitutedmay be the same base (e.g., all A or all C or all T or all G), twodifferent bases (e.g., A and C, A and T, or C and T), three differentbases (e.g., A, C and T or A, C and T) or at least four different bases.As a non-limiting example, the guanine base upstream of the codingregion in the oncology-related primary construct may be substituted withadenine, cytosine, thymine, or any of the nucleotides described herein.In another non-limiting example the substitution of guanine bases in theoncology-related primary construct may be designed so as to leave oneguanine base in the region downstream of the transcription start siteand before the start codon (see Esvelt et al. Nature (2011)472(7344):499-503; herein incorporated by reference in its entirety). Asa non-limiting example, at least 5 nucleotides may be inserted at 1location downstream of the transcription start site but upstream of thestart codon and the at least 5 nucleotides may be the same base type.

cDNA Template Removal and Clean-Up

The cDNA template may be removed using methods known in the art such as,but not limited to, treatment with Deoxyribonuclease I (DNase I). RNAclean-up may also include a purification method such as, but not limitedto, AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, Mass.),HPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

Capping and/or Tailing Reactions

The oncology-related primary construct or oncology-related mmRNA mayalso undergo capping and/or tailing reactions. A capping reaction may beperformed by methods known in the art to add a 5′ cap to the 5′ end ofthe oncology-related primary construct. Methods for capping include, butare not limited to, using a Vaccinia Capping enzyme (New EnglandBiolabs, Ipswich, Mass.).

A poly-A tailing reaction may be performed by methods known in the art,such as, but not limited to, 2′ O-methyltransferase and by methods asdescribed herein. If the oncology-related primary construct generatedfrom cDNA does not include a poly-T, it may be beneficial to perform thepoly-A-tailing reaction before the oncology-related primary construct iscleaned.

mRNA Purification

Primary construct or mmRNA purification may include, but is not limitedto, mRNA or mmRNA clean-up, quality assurance and quality control. mRNAor mmRNA clean-up may be performed by methods known in the arts such as,but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers,Mass.), poly-T beads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek,Denmark) or HPLC based purification methods such as, but not limited to,strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term“purified” when used in relation to a polynucleotide such as a “purifiedmRNA or mmRNA” refers to one that is separated from at least onecontaminant. As used herein, a “contaminant” is any substance whichmakes another unfit, impure or inferior. Thus, a purifiedoncology-related polynucleotide (e.g., DNA and RNA) is present in a formor setting different from that in which it is found in nature, or a formor setting different from that which existed prior to subjecting it to atreatment or purification method.

A quality assurance and/or quality control check may be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In another embodiment, the oncology-related mRNA or oncology-relatedmmRNA may be sequenced by methods including, but not limited toreverse-transcriptase-PCR.

In one embodiment, the oncology-related mRNA or oncology-related mmRNAmay be quantified using methods such as, but not limited to, ultravioletvisible spectroscopy (UV/Vis). A non-limiting example of a UV/Visspectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, Mass.).The quantified oncology-related mRNA or oncology-related mmRNA may beanalyzed in order to determine if the oncology-related mRNA oroncology-related mmRNA may be of proper size, check that no degradationof the oncology-related mRNA or oncology-related mmRNA has occurred.Degradation of the oncology-related mRNA and/or oncology-related mmRNAmay be checked by methods such as, but not limited to, agarose gelelectrophoresis, HPLC based purification methods such as, but notlimited to, strong anion exchange HPLC, weak anion exchange HPLC,reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC(HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillaryelectrophoresis (CE) and capillary gel electrophoresis (CGE).

Signal Sequences

The oncology-related primary constructs or oncology-related mmRNA mayalso encode additional features which facilitate trafficking of theoncology-related polypeptides to therapeutically relevant sites. Onesuch feature which aids in protein trafficking is the signal sequence.As used herein, a “signal sequence” or “signal peptide” is apolynucleotide or polypeptide, respectively, which is from about 9 to200 nucleotides (3-60 amino acids) in length which is incorporated atthe 5′ (or N-terminus) of the coding region or polypeptide encoded,respectively. Addition of these sequences result in trafficking of theencoded oncology-related polypeptide to the endoplasmic reticulumthrough one or more secretory pathways. Some signal peptides are cleavedfrom the protein by signal peptidase after the proteins are transported.

Table 5 is a representative listing of protein signal sequences whichmay be incorporated for encoding by the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA of the invention.

TABLE 5 Signal Sequences NUCLEOTIDE SEQ SEQ SEQUENCE ID ENCODED ID IDDescription (5′-3′) NO. PEPTIDE NO. SS- α-1- ATGATGCCATCCTCAGT 32MMPSSVSWGIL  94 001 antitrypsin CTCATGGGGTATTTTGC LAGLCCLVPVSTCTTGGCGGGTCTGTGC LA TGTCTCGTGCCGGTGTC GCTCGCA SS- G-CSFATGGCCGGACCGGCGA 33 MAGPATQSPM  95 002 CTCAGTCGCCCATGAAA KLMALQLLLWCTCATGGCCCTGCAGTT HSALWTVQEA GTTGCTTTGGCACTCAG CCCTCTGGACCGTCCAA GAGGCGSS- Factor IX ATGCAGAGAGTGAACA 34 MQRVNMIMAE  96 003 TGATTATGGCCGAGTCCSPSLITICLLGYL CCATCGCTCATCACAAT LSAECTVFLDH CTGCCTGCTTGGTAC  ENANKILNRPKGCTTTCCGCCGAATGCA R CTGTCTTTCTGGATCAC GAGAATGCGAATAAGA TCTTGAACCGACCCAAACGG SS- Prolactin ATGAAAGGATCATTGCT 35 MKGSLLLLLVS  97 004GTTGCTCCTCGTGTCGA NLLLCQSVAP ACCTTCTGCTTTGCCAG TCCGTAGCCCCC SS- AlbuminATGAAATGGGTGACGTT 36 MKWVTFISLLF  98 005 CATCTCACTGTTGTTTTT LFSSAYSRGGTTCTCGTCCGCCTACT VFRR CCAGGGGAGTATTCCGC CGA SS- HMMSP38ATGTGGTGGCGGCTCTG 37 MWWRLWWLL  99 006 GTGGCTGCTCCTGTTGC LLLLLLPMWATCCTCTTGCTGTGGCCC ATGGTGTGGGCA MLS- ornithine TGCTCTTTAACCTCCGC 38MLFNLRILLNN 100 001 carbamoyltr ATCCTGTTGAATAACGC AAFRNGHNFM ansferaseTGCGTTCCGAAATGGGC VRNFRCGQPLQ ATAACTTCATGGTACGC AACTTCAGATGCGGCCAGCCACTCCAG MLS- Cytochrome ATGTCCGTCTTGACACC 39 MSVLTPLLLRG 101 002C Oxidase CCTGCTCTTGAGAGGGC LTGSARRLPVP subunit 8A TGACGGGGTCCGCTAGRAKIHSL ACGCCTGCCGGTACCGC GAGCGAAGATCCACTC CCTG MLS- CytochromeATGAGCGTGCTCACTCC 40 MSVLTPLLLRG 102 003 C Oxidase GTTGCTTCTTCGAGGGCLTGSARRLPVP subunit 8A TTACGGGATCGGCTCGG RAKIHSL AGGTTGCCCGTCCCGAGAGCGAAGATCCATTCGT TG SS- Type III, TGACAAAAATAACTTTA 41 MVTKITLSPQN 103007 bacterial TCTCCCCAGAATTTTAG FRIQKQETTLLK AATCCAAAAACAGGAAEKSTEKNSLAK ACCACACTACTAAAAG SILAVKNHFIEL AAAAATCAACCGAGAA RSKLSERFISHKAAATTCTTTAGCAAAAA NT GTATTCTCGCAGTAAAA ATCACTTCATCGAATTAAGGTCAAAATTATCGG AACGTTTTATTTCGCAT AAGAACACT SS- Viral ATGCTGAGCTTTGTGGA42 MLSFVDTRTLL 104 008 TACCCGCACCCTGCTGC LLAVTSCLATC TGCTGGCGGTGACCAGC QTGCCTGGCGACCTGCCA G SS- viral ATGGGCAGCAGCCAGG 43 MGSSQAPRMGS 105 009CGCCGCGCATGGGCAG VGGHGLMALL CGTGGGCGGCCATGGC MAGLILPGILACTGATGGCGCTGCTGAT GGCGGGCCTGATTCTGC CGGGCATTCTGGCG SS- ViralATGGCGGGCATTTTTTA 44 MAGIFYFLFSFL 106 010 TTTTCTGTTTAGCTTTCT FGICDGTTTGGCATTTGCGAT SS- Viral ATGGAAAACCGCCTGCT 45 MENRLLRVFLV 107 011GCGCGTGTTTCTGGTGT WAALTMDGAS GGGCGGCGCTGACCAT A GGATGGCGCGAGCGCG SS-Viral ATGGCGCGCCAGGGCT 46 MARQGCFGSY 108 012 GCTTTGGCAGCTATCAGQVISLFTFAIGV GTGATTAGCCTGTTTAC NLCLG CTTTGCGATTGGCGTGA ACCTGTGCCTGGGCSS- Bacillus ATGAGCCGCCTGCCGGT 47 MSRLPVLLLLQ 109 013 GCTGCTGCTGCTGCAGCLLVRPGLQ TGCTGGTGCGCCCGGGC CTGCAG SS- Bacillus ATGAAACAGCAGAAAC 48MKQQKRLYAR 110 014 GCCTGTATGCGCGCCTG LLTLLFALIFLL CTGACCCTGCTGTTTGCPHSSASA GCTGATTTTTCTGCTGC CGCATAGCAGCGCGAG CGCG SS- SecretionATGGCGACGCCGCTGCC 49 MATPLPPPSPRH 111 015 signal TCCGCCCTCCCCGCGGCLRLLRLLLSG ACCTGCGGCTGCTGCGG CTGCTGCTCTCCGCCCT CGTCCTCGGC SS- SecretionATGAAGGCTCCGGGTC 50 MKAPGRLVLIIL 112 016 signal GGCTCGTGCTCATCATC CSVVFSCTGTGCTCCGTGGTCTT CTCT SS- Secretion ATGCTTCAGCTTTGGAA 51 MLQLWKLLCG 113017 signal ACTTGTTCTCCTGTGCG VLT GCGTGCTCACT SS- SecretionATGCTTTATCTCCAGGG 52 MLYLQGWSMP 114 018 signal TTGGAGCATGCCTGCTG AVATGGCA SS- Secretion ATGGATAACGTGCAGC 53 MDNVQPKIKHR 115 019 signalCGAAAATAAAACATCG PFCFSVKGHVK CCCCTTCTGCTTCAGTG MLRLDIINSLVTTGAAAGGCCACGTGAA TVFMLIVSVLA GATGCTGCGGCTGGATA LIP TTATCAACTCACTGGTAACAACAGTATTCATGCT CATCGTATCTGTGTTGG CACTGATACCA SS- SecretionATGCCCTGCCTAGACCA 54 MPCLDQQLTVH 116 020 signal ACAGCTCACTGTTCATGALPCPAQPSSLA CCCTACCCTGCCCTGCC FCQVGFLTA CAGCCCTCCTCTCTGGCCTTCTGCCAAGTGGGGT TCTTAACAGCA SS- Secretion ATGAAAACCTTGTTCAA 55MKTLFNPAPAI 117 021 signal TCCAGCCCCTGCCATTG ADLDPQFYTLSCTGACCTGGATCCCCAG DVFCCNESEAEI TTCTACACCCTCTCAGA LTGLTVGSAADTGTGTTCTGCTGCAATG A AAAGTGAGGCTGAGAT TTTAACTGGCCTCACGG TGGGCAGCGCTGCAGATGCT SS- Secretion ATGAAGCCTCTCCTTGT 56 MKPLLVVFVFL 118 022 signalTGTGTTTGTCTTTCTTTT FLWDPVLA CCTTTGGGATCCAGTGC TGGCA SS- SecretionATGTCCTGTTCCCTAAA 57 MSCSLKFTLIVI 119 023 signal GTTTACTTTGATTGTAAFFTCTLSSS TTTTTTTTTACTGTTGGC TTTCATCCAGC SS- Secretion ATGGTTCTTACTAAACC58 MVLTKPLQRNG 120 024 signal TCTTCAAAGAAATGGCA SMMSFENVKEKGCATGATGAGCTTTGAA SREGGPHAHTP AATGTGAAAGAAAAGA EEELCFVVTHTGCAGAGAAGGAGGGCC PQVQTTLNLFF CCATGCACACACACCCG HIFKVLTQPLSLAAGAAGAATTGTGTTTC LWG GTGGTAACACACTACCC TCAGGTTCAGACCACACTCAACCTGTTTTTCCAT ATATTCAAGGTTCTTAC TCAACCACTTTCCCTTC TGTGGGGT SS-Secretion ATGGCCACCCCGCCATT 59 MATPPFRLIRK 121 025 signalCCGGCTGATAAGGAAG MFSFKVSRWM ATGTTTTCCTTCAAGGT GLACFRSLAASGAGCAGATGGATGGGG CTTGCCTGCTTCCGGTC CCTGGCGGCATCC SS- SecretionATGAGCTTTTTCCAACT 60 MSFFQLLMKRK 122 026 signal CCTGATGAAAAGGAAGELIPLVVFMTV GAACTCATTCCCTTGGT AAGGASS GGTGTTCATGACTGTGG CGGCGGGTGGAGCCTCATCT SS- Secretion ATGGTCTCAGCTCTGCG 61 MVSALRGAPLI 123 027 signalGGGAGCACCCCTGATC RVHSSPVSSPSV AGGGTGCACTCAAGCC SGPAALVSCLSSCTGTTTCTTCTCCTTCTG QSSALS TGAGTGGACCACGGAG GCTGGTGAGCTGCCTGTCATCCCAAAGCTCAGCT CTGAGC SS- Secretion ATGATGGGGTCCCCAGT 62 MMGSPVSHLLA124 028 signal GAGTCATCTGCTGGCCG GFCVWVVLG GCTTCTGTGTGTGGGTC GTCTTGGGCSS- Secretion ATGGCAAGCATGGCTG 63 MASMAAVLTW 125 029 signalCCGTGCTCACCTGGGCT ALALLSAFSAT CTGGCTCTTCTTTCAGC QA GTTTTCGGCCACCCAGG CASS- Secretion ATGGTGCTCATGTGGAC 64 MVLMWTSGDA 126 030 signalCAGTGGTGACGCCTTCA FKTAYFLLKGA AGACGGCCTACTTCCTG PLQFSVCGLLQCTGAAGGGTGCCCCTCT VLVDLAILGQA GCAGTTCTCCGTGTGCG TA GCCTGCTGCAGGTGCTGGTGGACCTGGCCATCCT GGGGCAGGCCTACGCC SS- Secretion ATGGATTTTGTCGCTGG 65MDFVAGAIGGV 127 031 signal AGCCATCGGAGGCGTCT CGVAVGYPLDTGCGGTGTTGCTGTGGGC VKVRIQTEPLY TACCCCCTGGACACGGT TGIWHCVRDTYGAAGGTCAGGATCCAG HRERVWGFYR ACGGAGCCAAAGTACA GLSLPVCTVSLCAGGCATCTGGCACTGC VSS GTCCGGGATACGTATCA CCGAGAGCGCGTGTGG GGCTTCTACCGGGGCCTC TCGCTGCCCGTGTGCAC GGTGTCCCTGGTATCTT CC SS- SecretionATGGAGAAGCCCCTCTT 66 MEKPLFPLVPL 128 032 signal CCCATTAGTGCCTTTGCHWFGFGYTAL ATTGGTTTGGCTTTGGC VVSGGIVGYVK TACACAGCACTGGTTGT TGSVPSLAAGLTTCTGGTGGGATCGTTG LFGSLA GCTATGTAAAAACAGG CAGCGTGCCGTCCCTGGCTGCAGGGCTGCTCTTC GGCAGTCTAGCC SS- Secretion ATGGGTCTGCTCCTTCC 67MGLLLPLALCIL 129 033 signal CCTGGCACTCTGCATCC VLC TAGTCCTGTGC SS-Secretion ATGGGGATCCAGACGA 68 MGIQTSPVLLA 130 034 signalGCCCCGTCCTGCTGGCC SLGVGLVTLLG TCCCTGGGGGTGGGGCT LAVG GGTCACTCTGCTCGGCCTGGCTGTGGGC SS- Secretion ATGTCGGACCTGCTACT 69 MSDLLLLGLIG 131 035signal ACTGGGCCTGATTGGGG GLTLLLLLTLLA GCCTGACTCTCTTACTG FACTGCTGACGCTGCTAGC CTTTGCC SS- Secretion ATGGAGACTGTGGTGAT 70 METVVIVAIGV132 036 signal TGTTGCCATAGGTGTGC LATIFLASFAAL TGGCCACCATGTTTCTG VLVCRQGCTTCGTTTGCAGCCTT GGTGCTGGTTTGCAGGC AG SS- Secretion ATGCGCGGCTCTGTGGA71 MAGSVECTWG 133 037 signal GTGCACCTGGGGTTGGG WGHCAPSPLLLGGCACTGTGCCCCCAGC WTLLLFAAPFG CCCCTGCTCCTTTGGAC LLG TCTACTTCTGTTTGCAGCCCCATTTGGCCTGCTG GGG SS- Secretion ATGATGCCGTCCCGTAC 72 MMPSRTNLATG 134038 signal CAACCTGGCTACTGGAA IPSSKVKYSRLS TCCCCAGTAGTAAAGTG STDDGYIDLQFAAATATTCAAGGCTCTC KKTPPKIPYKAI CAGCACAGACGATGGC ALATVLFLIGATACATTGACCTTCAGTT TAAGAAAACCCCTCCTA AGATCCCTTATAAGGCC ATCGCACTTGCCACTGTGCTGTTTTTGATTGGCG CC SS- Secretion ATGGCCCTGCCCCAGAT 73 MALPQMCDGS 135039 signal GTGTGACGGGAGCCAC HLASTLRYCMT TTGGCCTCCACCCTCCG VSGTVVLVAGTCTATTGCATGACAGTCA LCFA GCGGCACAGTGGTTCTG GTGGCCGGGACGCTCTG CTTCGCT SS-Vrg-6 TGAAAAAGTGGTTCGTT 74 MKKWFVAAGI 136 041 GCTGCCGGCATCGGCGCGAGLLMLSSAA TGCCGGACTCATGCTCT CCAGCGCCGCCA SS- PhoA ATGAAACAGAGCACCA 75MKQSTIALALL 137 042 TTGCGCTGGCGCTGCTG PLLFTPVTKA CCGCTGCTGTTTACCCCGGTGACCAAAGCG SS- OmpA ATGAAAAAAACCGCGA 76 MKKTAIAIAVA 138 043TTGCGATTGCGGTGGCG LAGFATVAQA CTGGCGGGCTTTGCGAC CGTGGCGCAGGCG SS- STIATGAAAAAACTGATGC 77 MKKLMLAIFFS 139 044 TGGCGATTTTTTTTAGC VLSFPSFSQSGTGCTGAGCTTTCCGAG CTTTAGCCAGAGC SS- STII ATGAAAAAAAACATTG 78 MKKNIAFLLAS140 045 CGTTTCTGCTGGCGAGC MFVFSIATNAY ATGTTTGTGTTTAGCAT ATGCGACCAACGCGTATG CG SS- Amylase ATGTTTGCGAAACGCTT 79 MFAKRFKTSLL 141046 TAAAACCAGCCTGCTGC PLFAGFLLLFHL CGCTGTTTGCGGGCTTT VLAGPAAASCTGCTGCTGTTTCATCT GGTGCTGGCGGGCCCG GCGGCGGCGAGC SS- AlphaATGCGCTTTCCGAGCAT 80 MRFPSIFTAVLF 142 047 Factor TTTTACCGCGGTGCTGTAASSALA TTGCGGCGAGCAGCGC GCTGGCG SS- Alpha ATGCGCTTTCCGAGCAT 81MRFPSIFTTVLF 143 048 Factor TTTTACCACCGTGCTGT AASSALA TTGCGGCGAGCAGCGCGCTGGCG SS- Alpha ATGCGCTTTCCGAGCAT 82 MRFPSIFTSVLF 144 049 FactorTTTTACCAGCGTGCTGT AASSALA TTGCGGCGAGCAGCGC GCTGGCG SS- AlphaATGCGCTTTCCGAGCAT 83 MRFPSIFTHVLF 145 050 Factor TTTTACCCATGTGCTGTAASSALA TTGCGGCGAGCAGCGC GCTGGCG SS- Alpha ATGCGCTTTCCGAGCAT 84MRFPSIFTIVLF 146 051 Factor TTTTACCATTGTGCTGT AASSALA TTGCGGCGAGCAGCGCGCTGGCG SS- Alpha ATGCGCTTTCCGAGCAT 85 MRFPSIFTFVLF 147 052 FactorTTTTACCTTTGTGCTGTT AASSALA TGCGGCGAGCAGCGCG CTGGCG SS- AlphaATGCGCTTTCCGAGCAT 86 MRFPSIFTEVLF 148 053 Factor TTTTACCGAAGTGCTGTAASSALA TTGCGGCGAGCAGCGC GCTGGCG SS- Alpha ATGCGCTTTCCGAGCAT 87MRFPSIFTGVLF 149 054 Factor TTTTACCGGCGTGCTGT AASSALA TTGCGGCGAGCAGCGCGCTGGCG SS- Endo- ATGCGTTCCTCCCCCCT 88 MRSSPLLRSAV 150 055 glucanase CCTCCGCTCCGCCGTTG VAALPVLALA V TGGCCGCCCTGCCGGTG TTGGCCCTTGCC SS-Secretion ATGGGCGCGGCGGCCG 89 MGAAAVRWHL 151 056 signalTGCGCTGGCACTTGTGC CVLLALGTRGR GTGCTGCTGGCCCTGGG L CACACGCGGGCGGCTG SS-Fungal ATGAGGAGCTCCCTTGT 90 MRSSLVLFFVS 152 057 GCTGTTCTTTGTCTCTG AWTALACGTGGACGGCCTTGGCC AG SS- Fibronectin ATGCTCAGGGGTCCGG 91 MLRGPGPGRLL 153058 GACCCGGGCGGCTGCT LLAVLCLGTSV GCTGCTAGCAGTCCTGT RCTETGKSKRGCCTGGGGACATCGGT GCGCTGCACCGAAACC GGGAAGAGCAAGAGG SS- FibronectinATGCTTAGGGGTCCGGG 92 MLRGPGPGLLL 154 059 GCCCGGGCTGCTGCTGC LAVQCLGTAVPTGGCCGTCCAGCTGGGG STGA ACAGCGGTGCCCTCCAC G SS- FibronectinATGCGCCGGGGGGCCC 93 MRRGALTGLLL 155 060 TGACCGGGCTGCTCCTG VLCLSVVLRAAGTCCTGTGCCTGAGTGT PSATSKKRR TGTGCTACGTGCAGCCC CCTCTGCAACAAGCAAGAAGCGCAGG

In the table, SS is secretion signal and MLS is mitochondrial leadersignal. The oncology-related primary constructs or oncology-relatedmmRNA of the present invention may be designed to encode any of thesignal sequences of SEQ ID NOs 94-155, or fragments or variants thereof.These sequences may be included at the beginning of the oncology-relatedpolypeptide coding region, in the middle or at the terminus oralternatively into a flanking region. Further, any of theoncology-related polynucleotide primary constructs of the presentinvention may also comprise one or more of the sequences defined by SEQID NOs 32-93. These may be in the first region or either flankingregion.

Additional signal sequences which may be utilized in the presentinvention include those taught in, for example, databases such as thosefound at www.signalpeptide.de/ or proline.bic.nus.edu.sg/spdb/. Thosedescribed in U.S. Pat. Nos. 8,124,379; 7,413,875 and 7,385,034 are alsowithin the scope of the invention and the contents of each areincorporated herein by reference in their entirety.

Target Selection

According to the present invention, the oncology-related primaryconstructs comprise at least a first region of linked nucleosidesencoding at least one oncology-related polypeptide of interest. Theoncology-related polypeptides of interest or “targets” oroncology-related proteins and oncology-related peptides of the presentinvention are listed in Lengthy Table 6 and Table 7. Shown inLengthyTable 6, in addition to the target number (Target No), name anddescription of the gene encoding the oncology-related polypeptide ofinterest (Target Description) are the ENSEMBL Transcript ID (ENST) andSEQ ID NO (Trans SEQ ID NO), the ENSEMBL Protein ID (ENSP) and SEQ ID NO(Peptide SEQ ID NO) and when available the optimized transcript and/orORF sequence identifier (Optim Trans SEQ ID NO or Optimized ORF SEQ IDNO). Shown in Table 7, are familiar cancer syndromes, tumor suppressorgenes and sequence identifiers (e.g., ENST SEQ ID for the sequenceidentifiers associated with the ENSEMBL transcript sequences or ENSP SEQID for the sequence identifiers associated with the ENSEMBL proteinsequences), function of the tumor suppressor gene, chromosomal location,tumor type observed. For any particular gene there may exist one or morevariants or isoforms. Where these exist, they are shown in the tables aswell. It will be appreciated by those of skill in the art that disclosedin the Tables are potential flanking regions. These are encoded in eachENST transcript either to the 5′ (upstream) or 3′ (downstream) of theORF or coding region. The coding region is definitively and specificallydisclosed by teaching the ENSP sequence. Consequently, the sequencestaught flanking that encoding the protein are considered flankingregions. It is also possible to further characterize the 5′ and 3′flanking regions by utilizing one or more available databases oralgorithms. Databases have annotated the features contained in theflanking regions of the ENST transcripts and these are available in theart.

TABLE 7 Familial Cancer Syndrome Targets Familial Tumor ENST ENSP CancerSuppressor SEQ SEQ Chromosomal Tumor Types Syndrome Gene ID ID FunctionLocation Observed Li-Fraumeni P53 — — cell cycle 17p13.1 brain tumors,Syndrome regulation, sarcomas, apoptosis leukemia, breast cancerFamilial RB1 3632-3634 8169-8171 cell cycle 13q14.1-q14.2retinoblastoma, Retinoblastoma regulation osteogenic sarcoma Wilms WT14530-4537 9067-9074 transcriptional 11p13 pediatric kidney Tumorregulation cancer, most common form of childhood solid tumorNeurofibromatosis NF1 2762-2767 7299-7304 catalysis of 17q11.2neurofibromas, Type 1 RAS sarcomas, inactivation gliomasNeurofibromatosis NF2 2768-2780 7305-7317 linkage of cell 22q12.2Schwann cell Type 2 membrane to tumors, actin astrocytomas, cytoskeletonmeningiomas, ependymonas Familial APC 233-238 4781-4786 signaling5q21-q22 colon cancer Adenomatous through Polyposis adhesion moleculesto nucleus Tuberous TSC1 4317-4322 8854-8859 forms 9q34 seizures, mentalsclerosis 1 complex with retardation, TSC2 protein, facial inhibitsangiofibromas signaling to downstream effectors of mTOR Tuberous TSC24323-4328 8860-8865 forms 16p13.3 benign growths sclerosis 2 complexwith (hamartomas) TSC1 protein, in many tissues, inhibits astrocytomas,signaling to rhabdomyosarcomas downstream effectors of mTOR Deleted inDPC4, also — — regulation of 18q21.1 pancreatic Pancreatic known asTGF-β/BMP carcinoma, Carcinoma SMAD4 signal colon cancer 4, Familialtransduction juvenile polyposis syndrome Deleted in DCC 1329-13325875-5878 transmembrane 18q21.3 colorectal Colorectal receptor cancerCarcinoma involved in axonal guidance via netrins Familial BRCA1  57-5935118-5141 functions in 17q21 breast and Breast transcription, ovariancancer Cancer DNA binding, transcription coupled DNA repair, homologousrecombination, chromosomal stability, ubiquitination of proteins, andcentrosome replication Familial BRCA2 594-596 5142-5144 transcriptional13q12.3 breast and Breast (FANCD1) regulation of ovarian cancer Cancergenes involved in DNA repair and homologous recombination Cowden PTEN3057 7594 phosphoinositide 10q23.3 gliomas, breast syndrome3-phosphatase, cancer, thyroid protein cancer, head & tyrosine necksquamous phosphatase carcinoma Peutz- STK11 3806-3807 8343-8344phosphorylates 19p13.3 hyperpigmentation, Jeghers (serine- and activatesmultiple Syndrome threonine AMP- hamartomatous (PJS) kinase 11)activated polyps, kinase colorectal, (AMPK), breast and AMPK ovariancancers involved in stress responses, lipid and glucose meatabolismHereditary MSH2 2607-2616 7144-7153 DNA 2p22-p21 colon cancerNonpolyposis mismatch Colon repair Cancer type 1, HNPCC1 Hereditary MLH12588-2599 7125-7136 DNA 3p21.3 colon cancer Nonpolyposis mismatch Colonrepair Cancer type 2, HNPCC2 Familial CDH1 658-661 5206-5209 cell-cell16q22.1 gastric cancer, diffuse-type adhesion lobular breast gastricprotein cancer cancer von Hippel- VHL 4486-4488 9023-9025 regulation of3p26-p25 renal cancers, Lindau transcription hemangioblastomas, Syndromeelongation pheochromocytoma, through retinal angioma activation of aubiquitin ligase complex Familial CDKN2A 704-723 5252-5271 p16INK4 9p21melanoma, Melanoma inhibits cell- pancreatic cycle kinases cancer,others CDK4 and CDK6; p14ARF binds the p53 stabilizing protein MDM2Gorlin PTCH 3000-3016 7547-7553 transmembrane 9q22.3 basal cell skinSyndrome: (e.g., receptor for carcinoma. Nevoid basal PTCH1, sonic cellPTCH2) hedgehog carcinoma (shh), syndrome involved in (NBCCS) earlydevelopment through repression of action of smoothened Multiple MEN12570-2584 7107-7121 intrastrand 11q13 parathyroid and Endocrine DNApituitary Neoplasia crosslink adenomas, islet Type 1 repair cell tumors,carcinoid

Protein Cleavage Signals and Sites

In one embodiment, the oncology-related polypeptides of the presentinvention may include at least one protein cleavage signal containing atleast one protein cleavage site. The protein cleavage site may belocated at the N-terminus, the C-terminus, at any space between the N-and the C-termini such as, but not limited to, half-way between the N-and C-termini, between the N-terminus and the half way point, betweenthe half way point and the C-terminus, and combinations thereof.

The oncology-related polypeptides of the present invention may include,but is not limited to, a proprotein convertase (or prohormoneconvertase), thrombin or Factor Xa protein cleavage signal. Proproteinconvertases are a family of nine proteinases, comprising seven basicamino acid-specific subtilisin-like serine proteinases related to yeastkexin, known as prohormone convertase 1/3 (PC1/3), PC2, furin, PC4,PC5/6, paired basic amino-acid cleaving enzyme 4 (PACE4) and PC7, andtwo other subtilases that cleave at non-basic residues, calledsubtilisin kexin isozyme 1 (SKI-1) and proprotein convertase subtilisinkexin 9 (PCSK9). Non-limiting examples of protein cleavage signal aminoacid sequences are listing in Table 8. In Table 8, “X” refers to anyamino acid, “n” may be 0, 2, 4 or 6 amino acids and “*” refers to theprotein cleavage site. In Table 8, SEQ ID NO: 33885 refers to when n=4and SEQ ID NO: 33886 refers to when n=6.

TABLE 8 Protein Cleavage Site Sequences Protein Amino Acid SEQCleavage Signal Cleavage Sequence ID NO Proprotein R-X-X-R* 33883convertase R-X-K/R-R* 33884 K/R-Xn-K/R* 33885 or 33886 ThrombinL-V-P-R*-G-S 33887 L-V-P-R* 33888 A/F/G/I/L/T/V/M- 33889A/F/G/I/L/T/V/W-P-R* Factor Xa I-E-G-R* 33890 I-D-G-R* 33891 A-E-G-R*33892 A/F/G/I/L/T/V/M-D/E-G-R* 33893

In one embodiment, the oncology-related primary constructs and theoncology-related mmRNA of the present invention may be engineered suchthat the oncology-related primary construct or oncology-related mmRNAcontains at least one encoded protein cleavage signal. The encodedprotein cleavage signal may be located before the start codon, after thestart codon, before the coding region, within the coding region such as,but not limited to, half way in the coding region, between the startcodon and the half way point, between the half way point and the stopcodon, after the coding region, before the stop codon, between two stopcodons, after the stop codon and combinations thereof.

In one embodiment, the oncology-related primary constructs oroncology-related mmRNA of the present invention may include at least oneencoded protein cleavage signal containing at least one protein cleavagesite. The encoded protein cleavage signal may include, but is notlimited to, a proprotein convertase (or prohormone convertase), thrombinand/or Factor Xa protein cleavage signal. One of skill in the art mayuse Table 1 above or other known methods to determine the appropriateencoded protein cleavage signal to include in the oncology-relatedprimary constructs or mmRNA of the present invention. For example,starting with the signal of Table 8 and considering the codons of Table1 one can design a signal for the oncology-related primary constructwhich can produce a protein signal in the resulting oncology-relatedpolypeptide.

In one embodiment, the oncology-related polypeptides of the presentinvention include at least one protein cleavage signal and/or site.

As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Pub. No.20090227660, herein incorporated by reference in their entireties, use afurin cleavage site to cleave the N-terminal methionine of GLP-1 in theexpression product from the Golgi apparatus of the cells. In oneembodiment, the polypeptides of the present invention include at leastone protein cleavage signal and/or site with the proviso that thepolypeptide is not GLP-1.

In one embodiment, the oncology-related primary constructs oroncology-related mmRNA of the present invention includes at least oneencoded protein cleavage signal and/or site.

In one embodiment, the oncology-related primary constructs oroncology-related mmRNA of the present invention includes at least oneencoded protein cleavage signal and/or site with the proviso that theoncology-related primary construct or oncology-related mmRNA does notencode GLP-1.

In one embodiment, the oncology-related primary constructs oroncology-related mmRNA of the present invention may include more thanone coding region. Where multiple coding regions are present in theoncology-related primary construct or oncology-related mmRNA of thepresent invention, the multiple coding regions may be separated byencoded protein cleavage sites. As a non-limiting example, theoncology-related primary construct or oncology-related mmRNA may besigned in an ordered pattern. On such pattern follows AXBY form where Aand B are coding regions which may be the same or different codingregions and/or may encode the same or different oncology-relatedpolypeptides, and X and Y are encoded protein cleavage signals which mayencode the same or different protein cleavage signals. A second suchpattern follows the form AXYBZ where A and B are coding regions whichmay be the same or different coding regions and/or may encode the sameor different oncology-related polypeptides, and X, Y and Z are encodedprotein cleavage signals which may encode the same or different proteincleavage signals. A third pattern follows the form ABXCY where A, B andC are coding regions which may be the same or different coding regionsand/or may encode the same or different oncology-related polypeptides,and X and Y are encoded protein cleavage signals which may encode thesame or different protein cleavage signals.

In one embodiment, the oncology-related polypeptides, oncology-relatedprimary constructs and oncology-related mmRNA can also contain sequencesthat encode protein cleavage sites so that the oncology-relatedpolypeptides, oncology-related primary constructs and oncology-relatedmmRNA can be released from a carrier region or a fusion partner bytreatment with a specific protease for said protein cleavage site.

In one embodiment, the oncology-related polypeptides, primary constructsand mmRNA of the present invention may include a sequence encoding the2A peptide. In one embodiment, this sequence may be used to separate thecoding region of two or more polypeptides of interest. As a non-limitingexample, the sequence encoding the 2A peptide may be between codingregion A and coding region B (A-2Apep-B). The presence of the 2A peptidewould result in the cleavage of one long protein into protein A, proteinB and the 2A peptide. Protein A and protein B may be the same ordifferent polypeptides of interest. In another embodiment, the 2Apeptide may be used in the oncology-related polynucleotides, primaryconstructs and/or mmRNA of the present invention to produce two, three,four, five, six, seven, eight, nine, ten or more proteins.

Incorporating Post Transcriptional Control Modulators

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA of the present invention may include at leastone post transcriptional control modulator. These post transcriptionalcontrol modulators may be, but are not limited to, small molecules,compounds and regulatory sequences. As a non-limiting example, posttranscriptional control may be achieved using small molecules identifiedby PTC Therapeutics Inc. (South Plainfield, N.J.) using their GEMS™(Gene Expression Modulation by Small-Moleclues) screening technology.

The post transcriptional control modulator may be a gene expressionmodulator which is screened by the method detailed in or a geneexpression modulator described in International Publication No.WO2006022712, herein incorporated by reference in its entirety. Methodsidentifying RNA regulatory sequences involved in translational controlare described in International Publication No. WO2004067728, hereinincorporated by reference in its entirety; methods identifying compoundsthat modulate untranslated region dependent expression of a gene aredescribed in International Publication No. WO2004065561, hereinincorporated by reference in its entirety.

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA of the present invention may include at leastone post transcriptional control modulator is located in the 5′ and/orthe 3′ untranslated region of the polynucleotides, primary constructsand/or mmRNA of the present invention

In another embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA of the present invention may include at leastone post transcription control modulator to modulate prematuretranslation termination. The post transcription control modulators maybe compounds described in or a compound found by methods outlined inInternational Publication Nso. WO2004010106, WO2006044456, WO2006044682,WO2006044503 and WO2006044505, each of which is herein incorporated byreference in its entirety. As a non-limiting example, the compound maybind to a region of the 28S ribosomal RNA in order to modulate prematuretranslation termination (See e.g., WO2004010106, herein incorporated byreference in its entirety).

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA of the present invention may include at leastone post transcription control modulator to alter protein expression. Asa non-limiting example, the expression of VEGF may be regulated usingthe compounds described in or a compound found by the methods describedin International Publication Nos. WO2005118857, WO2006065480,WO2006065479 and WO2006058088, each of which is herein incorporated byreference in its entirety.

The oncology-related polynucleotides, primary constructs and/or mmRNA ofthe present invention may include at least one post transcriptioncontrol modulator to control translation. In one embodiment, the posttranscription control modulator may be a RNA regulatory sequence. As anon-limiting example, the RNA regulatory sequence may be identified bythe methods described in International Publication No. WO2006071903,herein incorporated by reference in its entirety.

III. MODIFICATIONS

Herein, in an oncology-related polynucleotide (such as anoncology-related primary construct or an oncology-related mRNAmolecule), the terms “modification” or, as appropriate, “modified” referto modification with respect to A, G, U or C ribonucleotides. Generally,herein, these terms are not intended to refer to the ribonucleotidemodifications in naturally occurring 5′-terminal mRNA cap moieties. In apolypeptide, the term “modification” refers to a modification ascompared to the canonical set of 20 amino acids, moiety)

The modifications may be various distinct modifications. In someembodiments, the coding region, the flanking regions and/or the terminalregions may contain one, two, or more (optionally different) nucleosideor nucleotide modifications. In some embodiments, a modifiedoncology-related polynucleotide, oncology-related primary construct, oroncology-related mmRNA introduced to a cell may exhibit reduceddegradation in the cell, as compared to an unmodified oncology-relatedpolynucleotide, oncology-related primary construct, or oncology-relatedmmRNA.

The oncology-related polynucleotides, oncology-related primaryconstructs, and oncology-related mmRNA can include any usefulmodification, such as to the sugar, the nucleobase, or theinternucleoside linkage (e.g. to a linking phosphate/to a phosphodiesterlinkage/to the phosphodiester backbone). One or more atoms of apyrimidine nucleobase may be replaced or substituted with optionallysubstituted amino, optionally substituted thiol, optionally substitutedalkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). Incertain embodiments, modifications (e.g., one or more modifications) arepresent in each of the sugar and the internucleoside linkage.Modifications according to the present invention may be modifications ofribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threosenucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids(PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additionalmodifications are described herein.

As described herein, the oncology-related polynucleotides,oncology-related primary constructs, and oncology-related mmRNA of theinvention do not substantially induce an innate immune response of acell into which the mRNA is introduced. Features of an induced innateimmune response include 1) increased expression of pro-inflammatorycytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc, and/or3) termination or reduction in protein translation.

In certain embodiments, it may desirable to intracellularly degrade amodified nucleic acid molecule introduced into the cell. For example,degradation of a modified oncology-related nucleic acid molecule may bepreferable if precise timing of protein production is desired. Thus, insome embodiments, the invention provides a modified oncology-relatednucleic acid molecule containing a degradation domain, which is capableof being acted on in a directed manner within a cell. In another aspect,the present disclosure provides oncology-related polynucleotidescomprising a nucleoside or nucleotide that can disrupt the binding of amajor groove interacting, e.g. binding, partner with the polynucleotide(e.g., where the modified nucleotide has decreased binding affinity tomajor groove interacting partner, as compared to an unmodifiednucleotide).

The oncology-related polynucleotides, oncology-related primaryconstructs, and oncology-related mmRNA can optionally include otheragents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs,antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triplehelix formation, aptamers, vectors, etc.). In some embodiments, theoncology-related polynucleotides, oncology-related primary constructs,or oncology-related mmRNA may include one or more messenger RNAs (mRNAs)and one or more modified nucleoside or nucleotides (e.g., mmRNAmolecules). Details for these oncology-related polynucleotides,oncology-related primary constructs, and oncology-related mmRNA follow.

Oncology-Related Polynucleotides and Oncology-Related Primary Constructs

The oncology-related polynucleotides, primary constructs, and mmRNA ofthe invention includes a first region of linked nucleosides encoding apolypeptide of interest, a first flanking region located at the 5′terminus of the first region, and a second flanking region located atthe 3′ terminus of the first region.

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA (e.g., the first region, first flanking region, orsecond flanking region) includes n number of linked nucleosides havingFormula (Ia) or Formula (Ia-1):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

--- is a single bond or absent;

each of R^(1′), R^(2′), R^(1″), R^(2″), R¹, R², R³, R⁴, and R⁵ is,independently, if present, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, orabsent; wherein the combination of R³ with one or more of R^(1′),R^(1″), R^(2′), R^(2″), or R⁵ (e.g., the combination of R^(1′) and R³,the combination of R^(1″) and R³, the combination of R^(2′) and R³, thecombination of R^(2″) and R³, or the combination of R⁵ and R³) can jointogether to form optionally substituted alkylene or optionallysubstituted heteroalkylene and, taken together with the carbons to whichthey are attached, provide an optionally substituted heterocyclyl (e.g.,a bicyclic, tricyclic, or tetracyclic heterocyclyl); wherein thecombination of R⁵ with one or more of R^(1′), R^(1″), R^(2′), or R^(2″)(e.g., the combination of R^(1′) and R⁵, the combination of R^(1″) andR⁵, the combination of R^(2′) and R⁵, or the combination of R^(2″) andR⁵) can join together to form optionally substituted alkylene oroptionally substituted heteroalkylene and, taken together with thecarbons to which they are attached, provide an optionally substitutedheterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl);and wherein the combination of R⁴ and one or more of R^(1′), R^(1″),R^(2′), R^(2″), R³, or R⁵ can join together to form optionallysubstituted alkylene or optionally substituted heteroalkylene and, takentogether with the carbons to which they are attached, provide anoptionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl); each of m′ and m″ is, independently, aninteger from 0 to 3 (e.g., from 0 to 2, from 0 to 1, from 1 to 3, orfrom 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof), wherein the combination of B and R^(1′), the combination of Band R^(2′), the combination of B and R^(1″), or the combination of B andR^(2″) can, taken together with the carbons to which they are attached,optionally form a bicyclic group (e.g., a bicyclic heterocyclyl) orwherein the combination of B, R^(1″), and R³ or the combination of B,R^(2″), and R³ can optionally form a tricyclic or tetracyclic group(e.g., a tricyclic or tetracyclic heterocyclyl, such as in Formula(IIo)-(IIp) herein). In some embodiments, the oncology-relatedpolynucleotide, primary construct, or mmRNA includes a modified ribose.In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA (e.g., the first region, the first flanking region,or the second flanking region) includes n number of linked nucleosideshaving Formula (Ia-2)-(Ia-5) or a pharmaceutically acceptable salt orstereoisomer thereof.

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA (e.g., the first region, the first flanking region,or the second flanking region) includes n number of linked nucleosideshaving Formula (Ib) or Formula (Ib-1):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

--- is a single bond or absent;

each of R¹, R^(3′), R^(3″), and R⁴ is, independently, H, halo, hydroxy,optionally substituted alkyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionallysubstituted hydroxyalkoxy, optionally substituted amino, azido,optionally substituted aryl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, or absent; and wherein the combination of R¹ and R^(3′) orthe combination of R¹ and R^(3″) can be taken together to formoptionally substituted alkylene or optionally substituted heteroalkylene(e.g., to produce a locked nucleic acid);

each R⁵ is, independently, H, halo, hydroxy, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, or absent;

each of Y¹, Y², and Y³ is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted alkoxyalkoxy, or optionally substituted amino;

n is an integer from 1 to 100,000; and

B is a nucleobase.

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA (e.g., the first region, first flanking region, orsecond flanking region) includes n number of linked nucleosides havingFormula (Ic):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

--- is a single bond or absent;

each of B¹, B², and B³ is, independently, a nucleobase (e.g., a purine,a pyrimidine, or derivatives thereof, as described herein), H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl, wherein one and only one of B¹, B²,and B³ is a nucleobase;

each of R^(b1), R^(b2), R^(b3), R³, and R⁵ is, independently, H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl oroptionally substituted aminoalkynyl;

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

wherein the ring including U can include one or more double bonds.

In particular embodiments, the ring including U does not have a doublebond between U—CB³R^(b3) or between CB³R^(b3)—C^(B2)R^(b2).

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA (e.g., the first region, first flanking region, orsecond flanking region) includes n number of linked nucleosides havingFormula (Id):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

each R³ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl;

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, optionally substituted alkylene (e.g.,methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA (e.g., the first region, first flanking region, orsecond flanking region) includes n number of linked nucleosides havingFormula (Ie):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of U′ and U″ is, independently, O, S, N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(U) is,independently, H, halo, or optionally substituted alkyl;

each R⁶ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl;

each Y^(5′) is, independently, O, S, optionally substituted alkylene(e.g., methylene or ethylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA (e.g., the first region, first flanking region, orsecond flanking region) includes n number of linked nucleosides havingFormula (If) or (If-1):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of U′ and U″ is, independently, O, S, N, N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(U) is,independently, H, halo, or optionally substituted alkyl (e.g., U′ is Oand U″ is N);

--- is a single bond or absent;

each of R^(1″), R^(2′), R^(1″), R^(2″), R³, and R⁴ is, independently, H,halo, hydroxy, thiol, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted aminoalkoxy, optionallysubstituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,optionally substituted amino, azido, optionally substituted aryl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, or absent; and wherein thecombination of R^(1′) and R³, the combination of R^(1″) and R³, thecombination of R^(2′) and R³, or the combination of R^(2″) and R³ can betaken together to form optionally substituted alkylene or optionallysubstituted heteroalkylene (e.g., to produce a locked nucleic acid);each of m′ and m″ is, independently, an integer from 0 to 3 (e.g., from0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If), and(IIa)-(IIp)), the ring including U has one or two double bonds.

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), each of R¹, R^(1″), and R^(1″), ifpresent, is H. In further embodiments, each of R², R^(2′), and R^(2″),if present, is, independently, H, halo (e.g., fluoro), hydroxy,optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionallysubstituted alkoxyalkoxy. In particular embodiments, alkoxyalkoxy is—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl). In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′is C₁₋₆ alkyl.

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), each of R², R^(2′), and R^(2″), ifpresent, is H. In further embodiments, each of R¹, R^(1″), and R^(1″),if present, is, independently, H, halo (e.g., fluoro), hydroxy,optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionallysubstituted alkoxyalkoxy. In particular embodiments, alkoxyalkoxy is—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl). In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′is C₁₋₆ alkyl.

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), each of R³, R⁴, and R⁵ is, independently,H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl,optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionallysubstituted alkoxyalkoxy. In particular embodiments, R³ is H, R⁴ is H,R⁵ is H, or R³, R⁴, and R⁵ are all H. In particular embodiments, R³ isC₁₋₆ alkyl, R⁴ is C₁₋₆ alkyl, R⁵ is C₁₋₆ alkyl, or R³, R⁴, and R⁵ areall C₁₋₆ alkyl. In particular embodiments, R³ and R⁴ are both H, and R⁵is C₁₋₆ alkyl.

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), R³ and R⁵ join together to formoptionally substituted alkylene or optionally substituted heteroalkyleneand, taken together with the carbons to which they are attached, providean optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl, such as trans-3′,4′ analogs, wherein R³ and R⁵join together to form heteroalkylene (e.g.,—(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein each of b1, b2, and b3 are,independently, an integer from 0 to 3).

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), R³ and one or more of R^(1′), R^(1″),R^(2′), R^(2″), or R⁵ join together to form optionally substitutedalkylene or optionally substituted heteroalkylene and, taken togetherwith the carbons to which they are attached, provide an optionallysubstituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclicheterocyclyl, R³ and one or more of R^(1′), R^(1″), R^(2′), R^(2″), orR⁵ join together to form heteroalkylene (e.g.,—(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein each of b1, b2, and b3 are,independently, an integer from 0 to 3).

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), R⁵ and one or more of R^(1′), R^(1″),R^(2′), or R^(2″) join together to form optionally substituted alkyleneor optionally substituted heteroalkylene and, taken together with thecarbons to which they are attached, provide an optionally substitutedheterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl,R⁵ and one or more of R^(1′), R^(1″), R^(2′), or R^(2″) join together toform heteroalkylene (e.g., —(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, whereineach of b1, b2, and b3 are, independently, an integer from 0 to 3).

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), each Y² is, independently, 0, S, or—NR^(N1)—, wherein R^(N1) is H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, or optionallysubstituted aryl. In particular embodiments, Y² is NR^(N1)—, whereinR^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆ alkyl, such asmethyl, ethyl, isopropyl, or n-propyl).

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), each Y³ is, independently, O or S.

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), R¹ is H; each R² is, independently, H,halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g.,methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′ isC₁₋₆ alkyl); each Y² is, independently, O or —NR^(N1)—, wherein R^(N1)is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, or optionally substituted aryl (e.g.,wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆ alkyl,such as methyl, ethyl, isopropyl, or n-propyl)); and each Y³ is,independently, O or S (e.g., S). In further embodiments, R³ is H, halo(e.g., fluoro), hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy (e.g., methoxy or ethoxy), or optionally substitutedalkoxyalkoxy. In yet further embodiments, each Y¹ is, independently, Oor —NR^(N1)—, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl (e.g., wherein R^(N1) is H or optionallysubstituted alkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl,or n-propyl)); and each Y⁴ is, independently, H, hydroxy, thiol,optionally substituted alkyl, optionally substituted alkoxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino.

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), each R¹ is, independently, H, halo (e.g.,fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′ isC₁₋₆ alkyl); R² is H; each Y² is, independently, O or —NR^(N1)—, whereinR^(N1) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, or optionally substituted aryl(e.g., wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆alkyl, such as methyl, ethyl, isopropyl, or n-propyl)); and each Y³ is,independently, O or S (e.g., S). In further embodiments, R³ is H, halo(e.g., fluoro), hydroxy, optionally substituted alkyl, optionallysubstituted alkoxy (e.g., methoxy or ethoxy), or optionally substitutedalkoxyalkoxy. In yet further embodiments, each Y¹ is, independently, Oor —NR^(N1)—, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl (e.g., wherein R^(N1) is H or optionallysubstituted alkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl,or n-propyl)); and each Y⁴ is, independently, H, hydroxy, thiol,optionally substituted alkyl, optionally substituted alkoxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino.

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), the ring including U is in the β-D (e.g.,β-D-ribo) configuration.

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), the ring including U is in the α-L (e.g.,α-L-ribo) configuration.

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), one or more B is not pseudouridine (ψ) or5-methyl-cytidine (m⁵C). In some embodiments, about 10% to about 100% ofn number of B nucleobases is not ψ or m⁵C (e.g., from 10% to 20%, from10% to 35%, from 10% to 50%, from 10% to 60%, from 10% to 75%, from 10%to 90%, from 10% to 95%, from 10% to 98%, from 10% to 99%, from 20% to35%, from 20% to 50%, from 20% to 60%, from 20% to 75%, from 20% to 90%,from 20% to 95%, from 20% to 98%, from 20% to 99%, from 20% to 100%,from 50% to 60%, from 50% to 75%, from 50% to 90%, from 50% to 95%, from50% to 98%, from 50% to 99%, from 50% to 100%, from 75% to 90%, from 75%to 95%, from 75% to 98%, from 75% to 99%, and from 75% to 100% of nnumber of B is not ψ or m⁵C). In some embodiments, B is not ψ or m⁵C.

In some embodiments of the oncology-related polynucleotides, primaryconstructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr)), when B is an unmodified nucleobaseselected from cytosine, guanine, uracil and adenine, then at least oneof Y¹, Y², or Y³ is not O.

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA includes a modified ribose. In some embodiments, theoncology-related polynucleotide, primary construct, or mmRNA (e.g., thefirst region, the first flanking region, or the second flanking region)includes n number of linked nucleosides having Formula (IIa)-(IIc):

or a pharmaceutically acceptable salt or stereoisomer thereof. Inparticular embodiments, U is O or C(R^(U))_(nu), wherein nu is aninteger from 0 to 2 and each R^(U) is, independently, H, halo, oroptionally substituted alkyl (e.g., U is —CH₂— or —CH—). In otherembodiments, each of R¹, R², R³, R⁴, and R⁵ is, independently, H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, or absent (e.g., each R¹ and R² is,independently, H, halo, hydroxy, optionally substituted alkyl, oroptionally substituted alkoxy; each R³ and R⁴ is, independently, H oroptionally substituted alkyl; and R⁵ is H or hydroxy), and

is a single bond or double bond. In particular embodiments, thepolynucleotidesor mmRNA includes n number of linked nucleosides havingFormula (IIb-1)-(IIb-2):

or a pharmaceutically acceptable salt or stereoisomer thereof. In someembodiments, U is O or C(R^(U))_(nu), wherein nu is an integer from 0 to2 and each R^(U) is, independently, H, halo, or optionally substitutedalkyl (e.g., U is —CH₂— or —CH—). In other embodiments, each of R¹ andR² is, independently, H, halo, hydroxy, thiol, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, or absent(e.g., each R¹ and R² is, independently, H, halo, hydroxy, optionallysubstituted alkyl, or optionally substituted alkoxy, e.g., H, halo,hydroxy, alkyl, or alkoxy). In particular embodiments, R² is hydroxy oroptionally substituted alkoxy (e.g., methoxy, ethoxy, or any describedherein).

In particular embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA includes n number of linked nucleosides havingFormula (IIc-1)-(IIc-4):

or a pharmaceutically acceptable salt or stereoisomer thereof. In someembodiments, U is O or C(R^(U))_(nu), wherein nu is an integer from 0 to2 and each R^(U) is, independently, H, halo, or optionally substitutedalkyl (e.g., U is —CH₂— or —CH—). In some embodiments, each of R¹, R²,and R³ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, or absent(e.g., each R¹ and R² is, independently, H, halo, hydroxy, optionallysubstituted alkyl, or optionally substituted alkoxy, e.g., H, halo,hydroxy, alkyl, or alkoxy; and each R³ is, independently, H oroptionally substituted alkyl)). In particular embodiments, R² isoptionally substituted alkoxy (e.g., methoxy or ethoxy, or any describedherein). In particular embodiments, R¹ is optionally substituted alkyl,and R² is hydroxy. In other embodiments, R¹ is hydroxy, and R² isoptionally substituted alkyl. In further embodiments, R³ is optionallysubstituted alkyl.

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA includes an acyclic modified ribose. In someembodiments, the oncology-related polynucleotide, primary construct, ormmRNA (e.g., the first region, the first flanking region, or the secondflanking region) includes n number of linked nucleosides having Formula(IId)-(IIf):

or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA includes an acyclic modified hexitol. In someembodiments, the oncology-related polynucleotide, primary construct, ormmRNA (e.g., the first region, the first flanking region, or the secondflanking region) includes n number of linked nucleosides Formula(IIg)-(IIj):

or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA includes a sugar moiety having a contracted or anexpanded ribose ring. In some embodiments, the oncology-relatedpolynucleotide, primary construct, or mmRNA (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides having Formula (IIk)-(IIm):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of R^(1′), R^(1″), R^(2′), and R^(2″) is, independently, H, halo,hydroxy, optionally substituted alkyl, optionally substituted alkoxy,optionally substituted alkenyloxy, optionally substituted alkynyloxy,optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy,or absent; and wherein the combination of R^(2′) and R³ or thecombination of R^(2″) and R³ can be taken together to form optionallysubstituted alkylene or optionally substituted heteroalkylene.

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA includes a locked modified ribose. In someembodiments, the oncology-related polynucleotide, primary construct, ormmRNA (e.g., the first region, the first flanking region, or the secondflanking region) includes n number of linked nucleosides having Formula(IIn):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(3′) is O, S, or —NR^(N1)—, wherein R^(N1) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl and R^(3″) isoptionally substituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)or optionally substituted heteroalkylene (e.g., —CH₂NH—, —CH₂CH₂NH—,—CH₂OCH₂—, or —CH₂CH₂OCH₂—)(e.g., R^(3′) is O and R^(3″) is optionallysubstituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)).

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA includes n number of linked nucleosides havingFormula (IIn-1)-(II-n2):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(3′) is O, S, or —NR^(N1)—, wherein R^(N1) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl and R^(3″) isoptionally substituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)or optionally substituted heteroalkylene (e.g., —CH₂NH—, —CH₂CH₂NH—,—CH₂OCH₂—, or —CH₂CH₂OCH₂—) (e.g., R^(3′) is O and R^(3″) is optionallysubstituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)).

In some embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA includes a locked modified ribose that forms atetracyclic heterocyclyl. In some embodiments, the oncology-relatedpolynucleotide, primary construct, or mmRNA (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides having Formula (IIo):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(12a), R^(12c), T^(1′), T^(1″), T^(2′), T^(2″), V¹, and V³ are asdescribed herein.

Any of the formulas for the oncology-related polynucleotides, primaryconstructs, or mmRNA can include one or more nucleobases describedherein (e.g., Formulas (b1)-(b43)).

In one embodiment, the present invention provides methods of preparing apolynucleotide, primary construct, or mmRNA, wherein the polynucleotidecomprises n number of nucleosides having Formula (Ia), as definedherein:

the method comprising reacting a compound of Formula (IIIa), as definedherein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a polynucleotide, primary construct, or mmRNA comprising atleast one nucleotide (e.g., mmRNA molecule), the method comprising:

reacting a compound of Formula (IIIa), as defined herein, with a primer,a cDNA template, and an RNA polymerase.

In one embodiment, the present invention provides methods of preparing apolynucleotide, primary construct, or mmRNA comprising at least onenucleotide (e.g., mmRNA molecule), wherein the polynucleotide comprisesn number of nucleosides having Formula (Ia), as defined herein:

the method comprising reacting a compound of Formula (IIIa-1), asdefined herein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a polynucleotide, primary construct, or mmRNA comprising atleast one nucleotide (e.g., mmRNA molecule), the method comprising:

-   -   reacting a compound of Formula (IIIa-1), as defined herein, with        a primer, a cDNA template, and an RNA polymerase.

In one embodiment, the present invention provides methods of preparing amodified mRNA comprising at least one nucleotide (e.g., mmRNA molecule),wherein the polynucleotide comprises n number of nucleosides havingFormula (Ia-2), as defined herein:

the method comprising reacting a compound of Formula (IIIa-2), asdefined herein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a modified mRNA comprising at least one nucleotide (e.g.,mmRNA molecule), the method comprising:

reacting a compound of Formula (IIIa-2), as defined herein, with aprimer, a cDNA template, and an RNA polymerase.

In some embodiments, the reaction may be repeated from 1 to about 7,000times. In any of the embodiments herein, B may be a nucleobase ofFormula (b1)-(b43).

The oncology-related polynucleotides, primary constructs, and mmRNA canoptionally include 5′ and/or 3′ flanking regions, which are describedherein.

Modified RNA (mmRNA) Molecules

The present invention also includes building blocks, e.g., modifiedribonucleosides, modified ribonucleotides, of modified RNA (mmRNA)molecules. For example, these building blocks can be useful forpreparing the oncology-related polynucleotides, primary constructs, ormmRNA of the invention.

In some embodiments, the building block molecule has Formula (IIIa) or(IIIa-1):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinthe substituents are as described herein (e.g., for Formula (Ia) and(Ia-1)), and wherein when B is an unmodified nucleobase selected fromcytosine, guanine, uracil and adenine, then at least one of Y¹, Y², orY³ is not O.

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IVa)-(IVb):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, Formula (IVa) or (IVb) is combined with a modified uracil(e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), suchas formula (b1), (b8), (b28), (b29), or (b30)). In particularembodiments, Formula (IVa) or (IVb) is combined with a modified cytosine(e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36),such as formula (b10) or (b32)). In particular embodiments, Formula(IVa) or (IVb) is combined with a modified guanine (e.g., any one offormulas (b15)-(b17) and (b37)-(b40)). In particular embodiments,Formula (IVa) or (IVb) is combined with a modified adenine (e.g., anyone of formulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IVc)-(IVk):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IVc)-(IVk) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IVc)-(IVk) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IVc)-(IVk) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IVc)-(IVk) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (Va) or (Yb):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IXa)-(IXd):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXa)-(IXd) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXa)-(IXd) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXa)-(IXd) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXa)-(IXd) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IXe)-(IXg):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXe)-(IXg) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXe)-(IXg) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXe)-(IXg) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXe)-(IXg) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IXh)-(IXk):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXh)-(IXk) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXh)-(IXk) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXh)-(IXk) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXh)-(IXk) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IX1)-(IXr):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r1 and r2 is, independently, an integer from 0 to 5 (e.g., from 0to 3, from 1 to 3, or from 1 to 5) and B is as described herein (e.g.,any one of (b1)-(b43)). In particular embodiments, one of Formulas(IX1)-(IXr) is combined with a modified uracil (e.g., any one offormulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1),(b8), (b28), (b29), or (b30)). In particular embodiments, one ofFormulas (IX1)-(IXr) is combined with a modified cytosine (e.g., any oneof formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula(b10) or (b32)). In particular embodiments, one of Formulas (IX1)-(IXr)is combined with a modified guanine (e.g., any one of formulas(b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas(IX1)-(IXr) is combined with a modified adenine (e.g., any one offormulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can beselected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can beselected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5) and s1 is as described herein.

In some embodiments, the building block molecule, which may beincorporated into a nucleic acid (e.g., RNA, mRNA, polynucleotide,primary construct, or mmRNA), is a modified uridine (e.g., selected fromthe group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, is amodified cytidine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)). For example, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, is amodified adenosine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, is amodified guanosine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the chemical modification can include replacementof C group at C-5 of the ring (e.g., for a pyrimidine nucleoside, suchas cytosine or uracil) with N (e.g., replacement of the >CH group at C-5with >NR^(N1) group, wherein R^(N1) is H or optionally substitutedalkyl). For example, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In another embodiment, the chemical modification can include replacementof the hydrogen at C-5 of cytosine with halo (e.g., Br, Cl, F, or I) oroptionally substituted alkyl (e.g., methyl). For example, the buildingblock molecule, which may be incorporated into a polynucleotide, primaryconstruct, or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In yet a further embodiment, the chemical modification can include afused ring that is formed by the NH₂ at the C-4 position and the carbonatom at the C-5 position. For example, the building block molecule,which may be incorporated into a polynucleotide, primary construct, ormmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

Modifications on the Sugar

The modified nucleosides and nucleotides (e.g., building blockmolecules), which may be incorporated into a polynucleotide, primaryconstruct, or mmRNA (e.g., RNA or mRNA, as described herein), can bemodified on the sugar of the ribonucleic acid. For example, the 2′hydroxyl group (OH) can be modified or replaced with a number ofdifferent substituents. Exemplary substitutions at the 2′-positioninclude, but are not limited to, H, halo, optionally substituted C₁₋₆alkyl; optionally substituted C₁₋₆ alkoxy; optionally substituted C₆₋₁₀aryloxy; optionally substituted C₃₋₈ cycloalkyl; optionally substitutedC₃₋₈ cycloalkoxy; optionally substituted C₆₋₁₀ aryloxy; optionallysubstituted C₆₋₁₀ aryl-C₁₋₆ alkoxy, optionally substituted C₁₋₁₂(heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any describedherein); a polyethyleneglycol (PEG), —O(CH₂CH₂O)_(n)CH₂CH₂OR, where R isH or optionally substituted alkyl, and n is an integer from 0 to 20(e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4,from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA)in which the 2′-hydroxyl is connected by a C₁₋₆ alkylene or C₁₋₆heteroalkylene bridge to the 4′-carbon of the same ribose sugar, whereexemplary bridges included methylene, propylene, ether, or aminobridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein;amino as defined herein; and amino acid, as defined herein

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting modified nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino that also has a phosphoramidate backbone); multicyclic forms(e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA)(e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attachedto phosphodiester bonds), threose nucleic acid (TNA, where ribose isreplace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA,where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a polynucleotide, primaryconstruct, or mmRNA molecule can include nucleotides containing, e.g.,arabinose, as the sugar.

Modifications on the Nucleobase

The present disclosure provides for modified nucleosides andnucleotides. As described herein “nucleoside” is defined as a compoundcontaining a sugar molecule (e.g., a pentose or ribose) or a derivativethereof in combination with an organic base (e.g., a purine orpyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group. The modified nucleotides may bysynthesized by any useful method, as described herein (e.g., chemically,enzymatically, or recombinantly to include one or more modified ornon-natural nucleosides).

The modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil.

The modified nucleosides and nucleotides can include a modifiednucleobase. Examples of nucleobases found in RNA include, but are notlimited to, adenine, guanine, cytosine, and uracil. Examples ofnucleobase found in DNA include, but are not limited to, adenine,guanine, cytosine, and thymine. These nucleobases can be modified orwholly replaced to provide oncology-related polynucleotides, primaryconstructs, or mmRNA molecules having enhanced properties, e.g.,resistance to nucleases through disruption of the binding of a majorgroove binding partner. Table 9 below identifies the chemical faces ofeach canonical nucleotide. Circles identify the atoms comprising therespective chemical regions.

TABLE 9 Watson-Crick Major Groove Minor Groove Base-pairing Face FaceFace Pyrimi- dines Cytidine:

Uridine:

Purines Adenosine:

Guanosine:

In some embodiments, B is a modified uracil. Exemplary modified uracilsinclude those having Formula (b1)-(b5):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

is a single or double bond;

each of T^(1′), T^(1″), T^(2′), and T^(2″) is, independently, H,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy, or the combination of T^(1′) andT^(1″) or the combination of T^(2′) and T^(2″) join together (e.g., asin T²) to form O (oxo), S (thio), or Se (seleno);

each of V¹ and V² is, independently, O, S, N(R^(Vb))_(nv), orC(R^(Vb))_(nv), wherein nv is an integer from 0 to 2 and each R^(Vb) is,independently, H, halo, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,optionally substituted aminoalkyl (e.g., substituted with anN-protecting group, such as any described herein, e.g.,trifluoroacetyl), optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g.,substituted with an N-protecting group, such as any described herein,e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, or optionally substituted alkynyloxy (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl);

R¹⁰ is H, halo, optionally substituted amino acid, hydroxy, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aminoalkyl, optionallysubstituted hydroxyalkyl, optionally substituted hydroxyalkenyl,optionally substituted hydroxyalkynyl, optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted alkoxy, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,optionally substituted carboxyalkoxy, optionally substitutedcarboxyalkyl, or optionally substituted carbamoylalkyl;

R¹¹ is H or optionally substituted alkyl;

R^(12a) is H, optionally substituted alkyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, or optionally substitutedaminoalkynyl, optionally substituted carboxyalkyl (e.g., optionallysubstituted with hydroxy), optionally substituted carboxyalkoxy,optionally substituted carboxyaminoalkyl, or optionally substitutedcarbamoylalkyl; and

R^(12c) is H, halo, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, optionally substituted amino,optionally substituted hydroxyalkyl, optionally substitutedhydroxyalkenyl, optionally substituted hydroxyalkynyl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl.

Other exemplary modified uracils include those having Formula (b6)-(b9):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

is a single or double bond;

each of T^(1′), T^(1″), T^(2′), and T^(2″) is, independently, H,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy, or the combination of T^(1′) andT^(1″) join together (e.g., as in T¹) or the combination of T^(2′) andT^(2″) join together (e.g., as in T²) to form O (oxo), S (thio), or Se(seleno), or each T¹ and T² is, independently, O (oxo), S (thio), or Se(seleno);

each of W¹ and W² is, independently, N(R^(Wa))_(nw) or C(R^(Wa))_(nw),wherein nw is an integer from 0 to 2 and each R^(Wa) is, independently,H, optionally substituted alkyl, or optionally substituted alkoxy;

each V³ is, independently, O, S, N(R^(Va))_(nv), or C(R^(Va))_(nv),wherein nv is an integer from 0 to 2 and each R^(Wa) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted hydroxyalkyl, optionally substitutedhydroxyalkenyl, optionally substituted hydroxyalkynyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclyl, optionally substituted alkheterocyclyl,optionally substituted alkoxy, optionally substituted alkenyloxy, oroptionally substituted alkynyloxy, optionally substituted aminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted acylaminoalkyl (e.g., substituted with an N-protectinggroup, such as any described herein, e.g., trifluoroacetyl), optionallysubstituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl), and wherein R^(Va) and R^(12c)taken together with the carbon atoms to which they are attached can formoptionally substituted cycloalkyl, optionally substituted aryl, oroptionally substituted heterocyclyl (e.g., a 5- or 6-membered ring);

R^(12a) is H, optionally substituted alkyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, optionally substituted carboxyalkyl (e.g., optionallysubstituted with hydroxy and/or an O-protecting group), optionallysubstituted carboxyalkoxy, optionally substituted carboxyaminoalkyl,optionally substituted carbamoylalkyl, or absent;

R^(12b) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, optionally substituted alkaryl, optionally substitutedheterocyclyl, optionally substituted alkheterocyclyl, optionallysubstituted amino acid, optionally substituted alkoxycarbonylacyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedalkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl,optionally substituted alkoxycarbonylalkynyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl,

wherein the combination of R^(12b) and T¹ or the combination of R^(12b)and R^(12c) can join together to form optionally substitutedheterocyclyl; and

R^(12c) is H, halo, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, optionally substituted amino,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,or optionally substituted aminoalkynyl.

Further exemplary modified uracils include those having Formula(b28)-(b31):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T¹ and T² is, independently, O (oxo), S (thio), or Se (seleno);

each R^(Vb′) and R^(Vb″) is, independently, H, halo, optionallysubstituted amino acid, optionally substituted alkyl, optionallysubstituted haloalkyl, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted acylaminoalkyl (e.g., substituted with an N-protectinggroup, such as any described herein, e.g., trifluoroacetyl), optionallysubstituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl) (e.g., R^(Vb′) is optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted aminoalkyl, e.g., substituted with an N-protecting group,such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl);

R^(12a) is H, optionally substituted alkyl, optionally substitutedcarboxyaminoalkyl, optionally substituted aminoalkyl (e.g., e.g.,substituted with an N-protecting group, such as any described herein,e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, or optionally substituted aminoalkynyl; and

R^(12b) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substituted aminoalkynyl(e.g., e.g., substituted with an N-protecting group, such as anydescribed herein, e.g., trifluoroacetyl, or sulfoalkyl),

optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,optionally substituted carboxyalkoxy, optionally substitutedcarboxyalkyl, or optionally substituted carbamoylalkyl.

In particular embodiments, T¹ is O (oxo), and T² is S (thio) or Se(seleno). In other embodiments, T¹ is S (thio), and T² is O (oxo) or Se(seleno). In some embodiments, R^(Vb′) is H, optionally substitutedalkyl, or optionally substituted alkoxy.

In other embodiments, each R^(12a) and R^(12b) is, independently, H,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted hydroxyalkyl. Inparticular embodiments, R^(12a) is H. In other embodiments, both R^(12a)and R^(12b) are H.

In some embodiments, each R^(Vb′) of R^(12b) is, independently,optionally substituted aminoalkyl (e.g., substituted with anN-protecting group, such as any described herein, e.g., trifluoroacetyl,or sulfoalkyl), optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, or optionally substituted acylaminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl). In some embodiments, the amino and/oralkyl of the optionally substituted aminoalkyl is substituted with oneor more of optionally substituted alkyl, optionally substituted alkenyl,optionally substituted sulfoalkyl, optionally substituted carboxy (e.g.,substituted with an O-protecting group), optionally substituted hydroxy(e.g., substituted with an O-protecting group), optionally substitutedcarboxyalkyl (e.g., substituted with an O-protecting group), optionallysubstituted alkoxycarbonylalkyl (e.g., substituted with an O-protectinggroup), or N-protecting group. In some embodiments, optionallysubstituted aminoalkyl is substituted with an optionally substitutedsulfoalkyl or optionally substituted alkenyl. In particular embodiments,R^(12a) and R^(Vb″) are both H. In particular embodiments, T¹ is O(oxo), and T² is S (thio) or Se (seleno).

In some embodiments, R^(Vb′) is optionally substitutedalkoxycarbonylalkyl or optionally substituted carbamoylalkyl.

In particular embodiments, the optional substituent for R^(12a),R^(12b), —R^(12c), or R^(Va) is a polyethylene glycol group (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl); or an amino-polyethylene glycol group (e.g.,—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl).

In some embodiments, B is a modified cytosine. Exemplary modifiedcytosines include compounds of Formula (b10)-(b14):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T^(3′) and T^(3″) is, independently, H, optionally substitutedalkyl, optionally substituted alkoxy, or optionally substitutedthioalkoxy, or the combination of T^(3′) and T^(3″) join together (e.g.,as in T³) to form O (oxo), S (thio), or Se (seleno);

each V⁴ is, independently, O, S, N(R^(Vc))_(nv), or C(R^(Vc))_(nv),wherein nv is an integer from 0 to 2 and each R^(Vc) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionallysubstituted with any substituent described herein, such as thoseselected from (1)-(21) for alkyl), wherein the combination of R^(13b)and R^(Vc) can be taken together to form optionally substitutedheterocyclyl;

each V⁵ is, independently, N(R^(Vd))_(nv), or C(R^(Vd))_(nv), wherein nvis an integer from 0 to 2 and each R^(Vd) is, independently, H, halo,optionally substituted amino acid, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionallysubstituted with any substituent described herein, such as thoseselected from (1)-(21) for alkyl) (e.g., V⁵ is —CH or N);

each of R^(13a) and R^(13b) is, independently, H, optionally substitutedacyl, optionally substituted acyloxyalkyl, optionally substituted alkyl,or optionally substituted alkoxy, wherein the combination of R^(13b) andR¹⁴ can be taken together to form optionally substituted heterocyclyl;

each R¹⁴ is, independently, H, halo, hydroxy, thiol, optionallysubstituted acyl, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted hydroxyalkyl (e.g., substituted with an O-protecting group),optionally substituted hydroxyalkenyl, optionally substitutedhydroxyalkynyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedacyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H,alkyl, aryl, or phosphoryl), azido, optionally substituted aryl,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkyl; and

each of R¹⁵ and R¹⁶ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl.

Further exemplary modified cytosines include those having Formula(b32)-(b35):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T¹ and T³ is, independently, O (oxo), S (thio), or Se (seleno);

each of R^(13a) and R^(13b) is, independently, H, optionally substitutedacyl, optionally substituted acyloxyalkyl, optionally substituted alkyl,or optionally substituted alkoxy, wherein the combination of R^(13b) andR¹⁴ can be taken together to form optionally substituted heterocyclyl;

each R¹⁴ is, independently, H, halo, hydroxy, thiol, optionallysubstituted acyl, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted hydroxyalkyl (e.g., substituted with an O-protecting group),optionally substituted hydroxyalkenyl, optionally substitutedhydroxyalkynyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedacyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H,alkyl, aryl, or phosphoryl), azido, optionally substituted aryl,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, optionally substituted aminoalkyl (e.g., hydroxyalkyl,alkyl, alkenyl, or alkynyl), optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl; and

each of R¹⁵ and R¹⁶ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl (e.g.,R¹⁵ is H, and R¹⁶ is H or optionally substituted alkyl).

In some embodiments, R¹⁵ is H, and R¹⁶ is H or optionally substitutedalkyl. In particular embodiments, R¹⁴ is H, acyl, or hydroxyalkyl. Insome embodiments, R¹⁴ is halo. In some embodiments, both R¹⁴ and R¹⁵ areH. In some embodiments, both R¹⁵ and R¹⁶ are H. In some embodiments,each of R¹⁴ and R¹⁵ and R¹⁶ is H. In further embodiments, each ofR^(13a) and R^(13b) is independently, H or optionally substituted alkyl.

Further non-limiting examples of modified cytosines include compounds ofFormula (b36):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each R^(13b) is, independently, H, optionally substituted acyl,optionally substituted acyloxyalkyl, optionally substituted alkyl, oroptionally substituted alkoxy, wherein the combination of R^(13b) andR^(14b) can be taken together to form optionally substitutedheterocyclyl;

each R^(14a) and R^(14b) is, independently, H, halo, hydroxy, thiol,optionally substituted acyl, optionally substituted amino acid,optionally substituted alkyl, optionally substituted haloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted hydroxyalkyl (e.g., substituted with anO-protecting group), optionally substituted hydroxyalkenyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted aminoalkoxy, optionallysubstituted alkoxyalkoxy, optionally substituted acyloxyalkyl,optionally substituted amino (e.g., —NHR, wherein R is H, alkyl, aryl,phosphoryl, optionally substituted aminoalkyl, or optionally substitutedcarboxyaminoalkyl), azido, optionally substituted aryl, optionallysubstituted heterocyclyl, optionally substituted alkheterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,or optionally substituted aminoalkynyl; and

each of R¹⁵ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl.

In particular embodiments, R^(14b) is an optionally substituted aminoacid (e.g., optionally substituted lysine). In some embodiments, R^(14a)is H.

In some embodiments, B is a modified guanine. Exemplary modifiedguanines include compounds of Formula (b15)-(b17):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T^(4′), T^(4″), T^(5′), T^(5″), T^(6′), and T^(6″) is,independently, H, optionally substituted alkyl, or optionallysubstituted alkoxy, and wherein the combination of T^(4′) and T^(4″)(e.g., as in T⁴) or the combination of T^(5′) and T^(5″) (e.g., as inT⁵) or the combination of T^(6′) and T^(6″) (e.g., as in T⁶) jointogether form O (oxo), S (thio), or Se (seleno);

each of V⁵ and V⁶ is, independently, O, S, N(R^(Vd))_(nv), orC(R^(Vd))_(nv), wherein nv is an integer from 0 to 2 and each R^(Vd) is,independently, H, halo, thiol, optionally substituted amino acid, cyano,amidine, optionally substituted aminoalkyl, optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, or optionally substituted alkynyloxy (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl), optionally substitutedthioalkoxy, or optionally substituted amino; and

each of R¹⁷, R¹⁸, R^(19a), R^(19b), R²¹, R²², R²³, and R²⁴ isindependently, H, halo, thiol, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted thioalkoxy, optionally substituted amino, or optionallysubstituted amino acid.

Exemplary modified guanosines include compounds of Formula (b37)-(b40):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each of T^(4′) is, independently, H, optionally substituted alkyl, oroptionally substituted alkoxy, and each T⁴ is, independently, O (oxo), S(thio), or Se (seleno);

each of R¹⁸, R^(19a), R^(19b), and R²¹ is, independently, H, halo,thiol, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted thioalkoxy,optionally substituted amino, or optionally substituted amino acid.

In some embodiments, R¹⁸ is H or optionally substituted alkyl. Infurther embodiments, T⁴ is oxo. In some embodiments, each of R^(19a) andR^(19b) is, independently, H or optionally substituted alkyl.

In some embodiments, B is a modified adenine. Exemplary modifiedadenines include compounds of Formula (b18)-(b20):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each V⁷ is, independently, O, S, N(R^(Ve))_(nv), or C(R^(Ve))_(nv),wherein nv is an integer from 0 to 2 and each R^(Ve) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy, oroptionally substituted alkynyloxy (e.g., optionally substituted with anysubstituent described herein, such as those selected from (1)-(21) foralkyl);

each R²⁵ is, independently, H, halo, thiol, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted thioalkoxy, or optionally substituted amino;

each of R^(26a) and R^(26b) is, independently, H, optionally substitutedacyl, optionally substituted amino acid, optionally substitutedcarbamoylalkyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted alkoxy, orpolyethylene glycol group (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl); or an amino-polyethylene glycol group(e.g., —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 isan integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1)is, independently, hydrogen or optionally substituted C₁₋₆ alkyl);

each R²⁷ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted thioalkoxy or optionallysubstituted amino;

each R²⁸ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, or optionally substituted alkynyl; and

each R²⁹ is, independently, H, optionally substituted acyl, optionallysubstituted amino acid, optionally substituted carbamoylalkyl,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted alkoxy, or optionallysubstituted amino.

Exemplary modified adenines include compounds of Formula (b41)-(b43):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

each R²⁵ is, independently, H, halo, thiol, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted thioalkoxy, or optionally substituted amino;

each of R^(26a) and R^(26b) is, independently, H, optionally substitutedacyl, optionally substituted amino acid, optionally substitutedcarbamoylalkyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted alkoxy, orpolyethylene glycol group (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl); or an amino-polyethylene glycol group(e.g., —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 isan integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1)is, independently, hydrogen or optionally substituted C₁₋₆ alkyl); and

each R²⁷ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted thioalkoxy, or optionallysubstituted amino.

In some embodiments, R^(26a) is H, and R^(26b) is optionally substitutedalkyl. In some embodiments, each of R^(26a) and R^(26b) is,independently, optionally substituted alkyl. In particular embodiments,R²⁷ is optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy. In other embodiments, R²⁵ isoptionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy.

In particular embodiments, the optional substituent for R^(26a),R^(26b), or R²⁹ is a polyethylene glycol group (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl); or an amino-polyethylene glycol group (e.g.,—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl).

In some embodiments, B may have Formula (b21):

wherein X¹² is, independently, O, S, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene, xa is aninteger from 0 to 3, and R^(12a) and T² are as described herein.

In some embodiments, B may have Formula (b22):

wherein R^(10′) is, independently, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, optionally substituted alkoxy,optionally substituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl, or optionallysubstituted carbamoylalkyl, and R¹¹, R^(12a), T¹ and T² are as describedherein.

In some embodiments, B may have Formula (b23):

wherein R¹⁰ is optionally substituted heterocyclyl (e.g., optionallysubstituted furyl, optionally substituted thienyl, or optionallysubstituted pyrrolyl), optionally substituted aryl (e.g., optionallysubstituted phenyl or optionally substituted naphthyl), or anysubstituent described herein (e.g., for R¹⁰); and wherein R¹¹ (e.g., Hor any substituent described herein), R^(12a) (e.g., H or anysubstituent described herein), T¹ (e.g., oxo or any substituentdescribed herein), and T² (e.g., oxo or any substituent describedherein) are as described herein.

In some embodiments, B may have Formula (b24):

wherein R^(14′) is, independently, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted alkaryl, optionally substituted alkheterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, optionally substituted alkoxy,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl, or optionallysubstituted carbamoylalkyl, and R^(13a), R^(13b), R¹⁵, and T³ are asdescribed herein.

In some embodiments, B may have Formula (b25):

wherein R^(14′) is optionally substituted heterocyclyl (e.g., optionallysubstituted furyl, optionally substituted thienyl, or optionallysubstituted pyrrolyl), optionally substituted aryl (e.g., optionallysubstituted phenyl or optionally substituted naphthyl), or anysubstituent described herein (e.g., for R¹⁴ or R^(14′)); and whereinR^(13a) (e.g., H or any substituent described herein), R^(13b) (e.g., Hor any substituent described herein), R¹⁵ (e.g., H or any substituentdescribed herein), and T³ (e.g., oxo or any substituent describedherein) are as described herein.

In some embodiments, B is a nucleobase selected from the groupconsisting of cytosine, guanine, adenine, and uracil. In someembodiments, B may be:

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm⁵s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e.,having the nucleobase deoxythymine), 1-methylpseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine (also known as1-methylpseudouridine (m¹ψ)), 3-(3-amino-3-carboxypropyl)uridine(acp³U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyladenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-0H-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o_(2y)W), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m^(2,7)G),N2,N2,7-dimethyl-guanosine(m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂ Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m′Im), and 2′-O-ribosylguanosine(phosphate) (Gr(p)).

The nucleobase of the nucleotide can be independently selected from apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can each be independently selected from adenine, cytosine,guanine, uracil, or hypoxanthine. In another embodiment, the nucleobasecan also include, for example, naturally-occurring and syntheticderivatives of a base, including pyrazolo[3,4-d]pyrimidines,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

Modifications on the Internucleoside Linkage

The modified nucleotides, which may be incorporated into apolynucleotide, primary construct, or mmRNA molecule, can be modified onthe internucleoside linkage (e.g., phosphate backbone). Herein, in thecontext of the polynucleotide backbone, the phrases “phosphate” and“phosphodiester” are used interchangeably. Backbone phosphate groups canbe modified by replacing one or more of the oxygen atoms with adifferent substituent. Further, the modified nucleosides and nucleotidescan include the wholesale replacement of an unmodified phosphate moietywith another internucleoside linkage as described herein. Examples ofmodified phosphate groups include, but are not limited to,phosphorothioate, phosphoroselenates, boranophosphates, boranophosphateesters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates,alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioateshave both non-linking oxygens replaced by sulfur. The phosphate linkercan also be modified by the replacement of a linking oxygen withnitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates),and carbon (bridged methylene-phosphonates).

The α-thio substituted phosphate moiety is provided to confer stabilityto RNA and DNA polymers through the unnatural phosphorothioate backbonelinkages. Phosphorothioate DNA and RNA have increased nucleaseresistance and subsequently a longer half-life in a cellularenvironment. Phosphorothioate linked oncology-related polynucleotides,primary constructs, or mmRNA molecules are expected to also reduce theinnate immune response through weaker binding/activation of cellularinnate immune molecules.

In specific embodiments, a modified nucleoside includes analpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine,5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine),5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or5′-O-(1-thiophosphate)-pseudouridine).

Other internucleoside linkages that may be employed according to thepresent invention, including internucleoside linkages which do notcontain a phosphorous atom, are described herein below.

Combinations of Modified Sugars, Nucleobases, and InternucleosideLinkages

The oncology-related polynucleotides, primary constructs, and mmRNA ofthe invention can include a combination of modifications to the sugar,the nucleobase, and/or the internucleoside linkage. These combinationscan include any one or more modifications described herein. Forexamples, any of the nucleotides described herein in Formulas (Ia),(Ia-1)-(Ia-3), (Ib)-(If), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr) can becombined with any of the nucleobases described herein (e.g., in Formulas(b1)-(b43) or any other described herein).

Synthesis of Oncology-Related Polypeptides, Primary Constructs, andmmRNA Molecules

The oncology-related polypeptides, oncology-related primary constructs,and oncology-related mmRNA molecules for use in accordance with theinvention may be prepared according to any useful technique, asdescribed herein. The modified nucleosides and nucleotides used in thesynthesis of oncology-related polynucleotides, oncology-related primaryconstructs, and oncology-related mmRNA molecules disclosed herein can beprepared from readily available starting materials using the followinggeneral methods and procedures. Where typical or preferred processconditions (e.g., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are provided, a skilled artisanwould be able to optimize and develop additional process conditions.Optimum reaction conditions may vary with the particular reactants orsolvent used, but such conditions can be determined by one skilled inthe art by routine optimization procedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry(e.g., UV-visible), or mass spectrometry, or by chromatography such ashigh performance liquid chromatography (HPLC) or thin layerchromatography.

Preparation of oncology-related polypeptides, oncology-related primaryconstructs, and oncology-related mmRNA molecules of the presentinvention can involve the protection and deprotection of variouschemical groups. The need for protection and deprotection, and theselection of appropriate protecting groups can be readily determined byone skilled in the art. The chemistry of protecting groups can be found,for example, in Greene, et al., Protective Groups in Organic Synthesis,2d. Ed., Wiley & Sons, 1991, which is incorporated herein by referencein its entirety.

The reactions of the processes described herein can be carried out insuitable solvents, which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of modified nucleosides and nucleotides(e.g., mmRNA molecules) can be carried out by any of numerous methodsknown in the art. An example method includes fractionalrecrystallization using a “chiral resolving acid” which is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, for example, optically activeacids, such as the D and L forms of tartaric acid, diacetyltartaricacid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid orthe various optically active camphorsulfonic acids. Resolution ofracemic mixtures can also be carried out by elution on a column packedwith an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

Modified nucleosides and nucleotides (e.g., building block molecules)can be prepared according to the synthetic methods described in Ogata etal., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568(1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each ofwhich are incorporated by reference in their entirety.

The oncology-related polypeptides, oncology-related primary constructs,and oncology-related mmRNA of the invention may or may not be uniformlymodified along the entire length of the molecule. For example, one ormore or all types of nucleotide (e.g., purine or pyrimidine, or any oneor more or all of A, G, U, C) may or may not be uniformly modified in anoncology-related polynucleotide of the invention, or in a givenpredetermined sequence region thereof (e.g. one or more of the sequenceregions represented in FIG. 1). In some embodiments, all nucleotides Xin an oncology-related polynucleotide of the invention (or in a givensequence region thereof) are modified, wherein X may any one ofnucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C,G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

Different sugar modifications, nucleotide modifications, and/orinternucleoside linkages (e.g., backbone structures) may exist atvarious positions in the oncology-related polynucleotide,oncology-related primary construct, or oncology-related mmRNA. One ofordinary skill in the art will appreciate that the nucleotide analogs orother modification(s) may be located at any position(s) of anoncology-related polynucleotide, oncology-related primary construct, oroncology-related mmRNA such that the function of the oncology-relatedpolynucleotide, oncology-related primary construct, or oncology-relatedmmRNA is not substantially decreased. A modification may also be a 5′ or3′ terminal modification. The oncology-related polynucleotide,oncology-related primary construct, or oncology-related mmRNA maycontain from about 1% to about 100% modified nucleotides (either inrelation to overall nucleotide content, or in relation to one or moretypes of nucleotide, i.e. any one or more of A, G, U or C) or anyintervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%,from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10%to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%,from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%,from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%,from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%,from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%,and from 95% to 100%).

In some embodiments, the oncology-related polynucleotide,oncology-related primary construct, or oncology-related mmRNA includes amodified pyrimidine (e.g., a modified uracil/uridine/U or modifiedcytosine/cytidine/C). In some embodiments, the uracil or uridine(generally: U) in the oncology-related polynucleotide, oncology-relatedprimary construct, or oncology-related mmRNA molecule may be replacedwith from about 1% to about 100% of a modified uracil or modifieduridine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1%to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%,from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10%to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100% of a modified uracil or modified uridine). The modified uracilor uridine can be replaced by a compound having a single uniquestructure or by a plurality of compounds having different structures(e.g., 2, 3, 4 or more unique structures, as described herein). In someembodiments, the cytosine or cytidine (generally: C) in theoncology-related polynucleotide, oncology-related primary construct, oroncology-related mmRNA molecule may be replaced with from about 1% toabout 100% of a modified cytosine or modified cytidine (e.g., from 1% to20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%,from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10%to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%,from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%,from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70%to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to95%, from 90% to 100%, and from 95% to 100% of a modified cytosine ormodified cytidine). The modified cytosine or cytidine can be replaced bya compound having a single unique structure or by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more uniquestructures, as described herein).

In some embodiments, the present disclosure provides methods ofsynthesizing an oncology-related polynucleotide, oncology-relatedprimary construct, or oncology-related mmRNA (e.g., the first region,first flanking region, or second flanking region) including n number oflinked nucleosides having Formula (Ia-1):

comprising:

a) reacting a nucleotide of Formula (IV-1):

with a phosphoramidite compound of Formula (V-1):

wherein Y⁹ is H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino,thiol, optionally substituted amino acid, or a peptide (e.g., includingfrom 2 to 12 amino acids);

and each P¹, P², and P³ is, independently, a suitable protecting group;and

denotes a solid support;

to provide an oncology-related polynucleotide, oncology-related primaryconstruct, or oncology-related mmRNA of Formula (VI-1):

and

b) oxidizing or sulfurizing the oncology-related polynucleotide,oncology-related primary construct, or oncology-related mmRNA of Formula(V) to yield an oncology-related polynucleotide, oncology-relatedprimary construct, or oncology-related mmRNA of Formula (VII-1):

and

c) removing the protecting groups to yield the oncology-relatedpolynucleotide, oncology-related primary construct, or oncology-relatedmmRNA of Formula (Ia).

In some embodiments, steps a) and b) are repeated from 1 to about 10,000times. In some embodiments, the methods further comprise a nucleotide(e.g., mmRNA molecule) selected from the group consisting of A, C, G andU adenosine, cytosine, guanosine, and uracil. In some embodiments, thenucleobase may be a pyrimidine or derivative thereof. In someembodiments, the oncology-related polynucleotide, oncology-relatedprimary construct, or oncology-related mmRNA is translatable.

Other components of oncology-related polynucleotides, oncology-relatedprimary constructs, and oncology-related mmRNA are optional, and arebeneficial in some embodiments. For example, a 5′ untranslated region(UTR) and/or a 3′UTR are provided, wherein either or both mayindependently contain one or more different nucleotide modifications. Insuch embodiments, nucleotide modifications may also be present in thetranslatable region. Also provided are oncology-related polynucleotides,oncology-related primary constructs, and oncology-related mmRNAcontaining a Kozak sequence.

Exemplary syntheses of modified nucleotides, which are incorporated intoan oncology-related modified nucleic acid or mmRNA, e.g., RNA or mRNA,are provided below in Scheme 1 through Scheme 11. Scheme 1 provides ageneral method for phosphorylation of nucleosides, including modifiednucleosides.

Various protecting groups may be used to control the reaction. Forexample, Scheme 2 provides the use of multiple protecting anddeprotecting steps to promote phosphorylation at the 5′ position of thesugar, rather than the 2′ and 3′ hydroxyl groups.

Modified nucleotides can be synthesized in any useful manner. Schemes 3,4, and 7 provide exemplary methods for synthesizing modified nucleotideshaving a modified purine nucleobase; and Schemes 5 and 6 provideexemplary methods for synthesizing modified nucleotides having amodified pseudouridine or pseudoisocytidine, respectively.

Schemes 8 and 9 provide exemplary syntheses of modified nucleotides.Scheme 10 provides a non-limiting biocatalytic method for producingnucleotides.

Scheme 11 provides an exemplary synthesis of a modified uracil, wherethe N1 position is modified with R12b, as provided elsewhere, and the5′-position of ribose is phosphorylated. T1, T2, R12a, R12b, and r areas provided herein. This synthesis, as well as optimized versionsthereof, can be used to modify other pyrimidine nucleobases and purinenucleobases (see e.g., Formulas (b1)-(b43)) and/or to install one ormore phosphate groups (e.g., at the 5′ position of the sugar). Thisalkylating reaction can also be used to include one or more optionallysubstituted alkyl group at any reactive group (e.g., amino group) in anynucleobase described herein (e.g., the amino groups in the Watson-Crickbase-pairing face for cytosine, uracil, adenine, and guanine).

Combinations of Nucleotides in mmRNA

Further examples of modified nucleotides and modified nucleotidecombinations are provided below in Table 10. These combinations ofmodified nucleotides can be used to form the oncology-relatedpolypeptides, oncology-related primary constructs, or oncology-relatedmmRNA of the invention.

Unless otherwise noted, the modified nucleotides may be completelysubstituted for the natural nucleotides of the modified nucleic acids ormmRNA of the invention. As a non-limiting example, the naturalnucleotide uridine may be substituted with a modified nucleosidedescribed herein. In another non-limiting example, the naturalnucleotide uridine may be partially substituted (e.g., about 0.1%, 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 99.9%) with at least one of the modifiednucleoside disclosed herein.

TABLE 10 Modified Nucleotide Modified Nucleotide Combinationα-thio-cytidine α-thio-cytidine/5-iodo-uridineα-thio-cytidine/N1-methyl-pseudouridine α-thio-cytidine/α-thio-uridineα-thio-cytidine/5-methyl-uridine α-thio-cytidine/pseudouridine about 50%of the cytosines are α-thio-cytidine pseudoisocytidinepseudoisocytidine/5-iodo-uridinepseudoisocytidine/N1-methyl-pseudouridinepseudoisocytidine/α-thio-uridine pseudoisocytidine/5-methyl-uridinepseudoisocytidine/pseudouridine about 25% of cytosines arepseudoisocytidine pseudoisocytidine/about 50% of uridines areN1-methyl-pseudouridine and about 50% of uridines are pseudouridinepseudoisocytidine/about 25% of uridines are N1-methyl-pseudouridine andabout 25% of uridines are pseudouridine pyrrolo-cytidinepyrrolo-cytidine/5-iodo-uridine pyrrolo-cytidine/N1-methyl-pseudouridinepyrrolo-cytidine/α-thio-uridine pyrrolo-cytidine/5-methyl-uridinepyrrolo-cytidine/pseudouridine about 50% of the cytosines arepyrrolo-cytidine 5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine5-methyl-cytidine/N1-methyl-pseudouridine5-methyl-cytidine/α-thio-uridine 5-methyl-cytidine/5-methyl-uridine5-methyl-cytidine/pseudouridine about 25% of cytosines are5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridinesare 2-thio-uridine about 50% of uridines are 5-methyl-cytidine/ about50% of uridines are 2-thio-uridine N4-acetyl-cytidineN4-acetyl-cytidine/5-iodo-uridineN4-acetyl-cytidine/N1-methyl-pseudouridineN4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridineN4-acetyl-cytidine/pseudouridine about 50% of cytosines areN4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidineN4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridineN4-acetyl-cytidine/2-thio-uridine about 50% of cytosines areN4-acetyl-cytidine/ about 50% of uridines are 2-thio-uridine

Further examples of modified nucleotide combinations are provided belowin Table 11. These combinations of modified nucleotides can be used toform the polypeptides, primary constructs, or mmRNA of the invention.

TABLE 11 Modified Nucleotide Modified Nucleotide Combination modifiedcytidine modified cytidine with (b10)/pseudouridine having one or moremodified cytidine with (b10)/N1-methyl- nucleobases of pseudouridineFormula (b10) modified cytidine with (b10)/5-methoxy-uridine modifiedcytidine with (b10)/5-methyl-uridine modified cytidine with(b10)/5-bromo-uridine modified cytidine with (b10)/2-thio-uridine about50% of cytidine substituted with modified cytidine (b10)/about 50% ofuridines are 2-thio- uridine modified cytidine modified cytidine with(b32)/pseudouridine having one or more modified cytidine with(b32)/N1-methyl- nucleobases of pseudouridine Formula (b32) modifiedcytidine with (b32)/5-methoxy-uridine modified cytidine with(b32)/5-methyl-uridine modified cytidine with (b32)/5-bromo-uridinemodified cytidine with (b32)/2-thio-uridine about 50% of cytidinesubstituted with modified cytidine (b32)/about 50% of uridines are2-thio- uridine modified uridine having modified uridine with(b1)/N4-acetyl-cytidine one or more nucleobases modified uridine with(b1)/5-methyl-cytidine of Formula (b1) modified uridine having modifieduridine with (b8)/N4-acetyl-cytidine one or more nucleobases modifieduridine with (b8)/5-methyl-cytidine of Formula (b8) modified uridinehaving modified uridine with (b28)/N4-acetyl-cytidine one or morenucleobases modified uridine with (b28)/5-methyl-cytidine of Formula(b28) modified uridine having modified uridine with(b29)/N4-acetyl-cytidine one or more nucleobases modified uridine with(b29)/5-methyl-cytidine of Formula (b29) modified uridine havingmodified uridine with (b30)/N4-acetyl-cytidine one or more nucleobasesmodified uridine with (b30)/5-methyl-cytidine of Formula (b30)

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14) (e.g., at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the uracils are replaced by acompound of Formula (b1)-(b9) (e.g., at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14), and at least 25% of the uracils arereplaced by a compound of Formula (b1)-(b9) (e.g., at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%).

IV. PHARMACEUTICAL COMPOSITIONS Formulation, Administration, Deliveryand Dosing

The present invention provides oncology-related polynucleotides,oncology-related primary constructs and oncology-related mmRNAcompositions and complexes in combination with one or morepharmaceutically acceptable excipients. Pharmaceutical compositions mayoptionally comprise one or more additional active substances, e.g.therapeutically and/or prophylactically active substances. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005(incorporated herein by reference).

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to oncology-relatedpolynucleotides, oncology-related primary constructs andoncology-related mmRNA to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

Formulations

The oncology-related polynucleotide, oncology-related primary construct,and oncology-related mmRNA of the invention can be formulated using oneor more excipients to: (1) increase stability; (2) increase celltransfection; (3) permit the sustained or delayed release (e.g., from adepot formulation of the oncology-related polynucleotide,oncology-related primary construct, or mmRNA); (4) alter thebiodistribution (e.g., target the oncology-related polynucleotide,oncology-related primary construct, or oncology-related mmRNA tospecific tissues or cell types); (5) increase the translation of encodedprotein in vivo; and/or (6) alter the release profile of encoded proteinin vivo. In addition to traditional excipients such as any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, excipients of thepresent invention can include, without limitation, lipidoids, liposomes,lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles,peptides, proteins, cells transfected with oncology-relatedpolynucleotide, primary oncology-related construct, or oncology-relatedmmRNA (e.g., for transplantation into a subject), hyaluronidase,nanoparticle mimics and combinations thereof. Further, theoncology-related polynucleotide, oncology-related primary construct, oroncology-related mmRNA of the present invention may be formulated usingself-assembled nucleic acid nanoparticles.

Accordingly, the formulations of the invention can include one or moreexcipients, each in an amount that together increases the stability ofthe oncology-related polynucleotide, oncology-related primary construct,or oncology-related mmRNA, increases cell transfection by theoncology-related polynucleotide, oncology-related primary construct, oroncology-related mmRNA, increases the expression of oncology-relatedpolynucleotide, oncology-related primary construct, or oncology-relatedmmRNA encoded protein, and/or alters the release profile ofoncology-related polynucleotide, oncology-related primary construct, oroncology-related mmRNA encoded proteins. Further, the oncology-relatedprimary construct and oncology-related mmRNA of the present inventionmay be formulated using self-assembled nucleic acid nanoparticles.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient may generally be equal to the dosage of theactive ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage including, but not limited to,one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient.

In some embodiments, the formulations described herein may contain atleast one oncology-related mmRNA. As a non-limiting example, theformulations may contain 1, 2, 3, 4 or 5 oncology-related mmRNA. In oneembodiment the formulation may contain modified mRNA encoding proteinsselected from categories such as, oncology-related proteins. In oneembodiment, the formulation contains at least three oncology-relatedmodified mRNA encoding oncology-related proteins. In one embodiment, theformulation contains at least five oncology-related modified mRNAencoding oncology-related proteins.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro,Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference in its entirety). The use of a conventionalexcipient medium may be contemplated within the scope of the presentdisclosure, except insofar as any conventional excipient medium may beincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition.

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter biological reaction such as, but not limited to,inflammation or may increase the biological effect of theoncology-related modified mRNA delivered to mammals.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, surface active agents and/or emulsifiers, preservatives,buffering agents, lubricating agents, and/or oils. Such excipients mayoptionally be included in the pharmaceutical formulations of theinvention.

Lipidoids

The synthesis of lipidoids has been extensively described andformulations containing these compounds are particularly suited fordelivery of oncology-related polynucleotides, primary constructs ormmRNA (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder etal., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 200826:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869;Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; all ofwhich are incorporated herein in their entireties).

While these lipidoids have been used to effectively deliver doublestranded small interfering RNA molecules in rodents and non-humanprimates (see Akinc et al., Nat Biotechnol. 2008 26:561-569;Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920;Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad SciUSA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 201129:1005-1010; all of which is incorporated herein in their entirety),the present disclosure describes their formulation and use in deliveringsingle stranded oncology-related polynucleotides, primary constructs, ormmRNA. Complexes, micelles, liposomes or particles can be preparedcontaining these lipidoids and therefore, can result in an effectivedelivery of the oncology-related polynucleotide, primary construct, ormmRNA, as judged by the production of an encoded protein, following theinjection of a lipidoid formulation via localized and/or systemic routesof administration. Lipidoid complexes of oncology-relatedpolynucleotides, primary constructs, or mmRNA can be administered byvarious means including, but not limited to, intravenous, intramuscular,or subcutaneous routes.

In vivo delivery of nucleic acids may be affected by many parameters,including, but not limited to, the formulation composition, nature ofparticle PEGylation, degree of loading, oligonucleotide to lipid ratio,and biophysical parameters such as, but not limited to, particle size(Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated byreference in its entirety). As an example, small changes in the anchorchain length of poly(ethylene glycol) (PEG) lipids may result insignificant effects on in vivo efficacy. Formulations with the differentlipidoids, including, but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry,401:61 (2010); herein incorporated by reference in its entirety),C12-200 (including derivatives and variants), and MD1, can be tested forin vivo activity.

The lipidoid referred to herein as “98N12-5” is disclosed by Akinc etal., Mol Ther. 2009 17:872-879 and is incorporated by reference in itsentirety. (See FIG. 2)

The lipidoid referred to herein as “C12-200” is disclosed by Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 (see FIG. 2) and Liu andHuang, Molecular Therapy. 2010 669-670 (see FIG. 2); both of which areherein incorporated by reference in their entirety. The lipidoidformulations can include particles comprising either 3 or 4 or morecomponents in addition to oncology-related polynucleotide, primaryconstruct, or mmRNA. As an example, formulations with certain lipidoids,include, but are not limited to, 98N12-5 and may contain 42% lipidoid,48% cholesterol and 10% PEG (C14 alkyl chain length). As anotherexample, formulations with certain lipidoids, include, but are notlimited to, C12-200 and may contain 50% lipidoid, 10%disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.

In one embodiment, a oncology-related polynucleotide, primary construct,or mmRNA formulated with a lipidoid for systemic intravenousadministration can target the liver. For example, a final optimizedintravenous formulation using a oncology-related polynucleotide, primaryconstruct, or mmRNA, and comprising a lipid molar composition of 42%98N12-5, 48% cholesterol, and 10% PEG-lipid with a final weight ratio ofabout 7.5 to 1 total lipid to oncology-related polynucleotide, primaryconstruct, or mmRNA, and a C14 alkyl chain length on the PEG lipid, witha mean particle size of roughly 50-60 nm, can result in the distributionof the formulation to be greater than 90% to the liver. (see, Akinc etal., Mol Ther. 2009 17:872-879; herein incorporated by reference in itsentirety). In another example, an intravenous formulation using aC12-200 (see U.S. provisional application 61/175,770 and publishedinternational application WO2010129709, each of which is hereinincorporated by reference in their entirety) lipidoid may have a molarratio of 50/10/38.5/1.5 of C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG, with a weight ratio of 7 to 1 total lipidto oncology-related polynucleotide, primary construct, or mmRNA, and amean particle size of 80 nm may be effective to deliver oncology-relatedpolynucleotide, primary construct, or mmRNA to hepatocytes (see, Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 herein incorporated byreference in its entirety). In another embodiment, an MD1lipidoid-containing formulation may be used to effectively deliveroncology-related polynucleotide, primary construct, or mmRNA tohepatocytes in vivo. The characteristics of optimized lipidoidformulations for intramuscular or subcutaneous routes may varysignificantly depending on the target cell type and the ability offormulations to diffuse through the extracellular matrix into the bloodstream. While a particle size of less than 150 nm may be desired foreffective hepatocyte delivery due to the size of the endothelialfenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879 hereinincorporated by reference in its entirety), use of a lipidoid-formulatedoncology-related polynucleotide, primary construct, or mmRNA to deliverthe formulation to other cells types including, but not limited to,endothelial cells, myeloid cells, and muscle cells may not be similarlysize-limited. Use of lipidoid formulations to deliver siRNA in vivo toother non-hepatocyte cells such as myeloid cells and endothelium hasbeen reported (see Akinc et al., Nat Biotechnol. 2008 26:561-569;Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; Cho et al. Adv.Funct. Mater. 2009 19:3112-3118; 8^(th) International Judah FolkmanConference, Cambridge, Mass. Oct. 8-9, 2010; each of which is hereinincorporated by reference in its entirety). Effective delivery tomyeloid cells, such as monocytes, lipidoid formulations may have asimilar component molar ratio. Different ratios of lipidoids and othercomponents including, but not limited to, disteroylphosphatidyl choline,cholesterol and PEG-DMG, may be used to optimize the formulation of theoncology-related polynucleotide, primary construct, or mmRNA fordelivery to different cell types including, but not limited to,hepatocytes, myeloid cells, muscle cells, etc. For example, thecomponent molar ratio may include, but is not limited to, 50% C12-200,10% disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG-DMG(see Leuschner et al., Nat Biotechnol 2011 29:1005-1010; hereinincorporated by reference in its entirety). The use of lipidoidformulations for the localized delivery of nucleic acids to cells (suchas, but not limited to, adipose cells and muscle cells) via eithersubcutaneous or intramuscular delivery, may not require all of theformulation components desired for systemic delivery, and as such maycomprise only the lipidoid and the oncology-related polynucleotide,primary construct, or mmRNA.

Combinations of different lipidoids may be used to improve the efficacyof oncology-related polynucleotide, primary construct, or mmRNA directedprotein production as the lipidoids may be able to increase celltransfection by the oncology-related polynucleotide, primary construct,or mmRNA; and/or increase the translation of encoded protein (seeWhitehead et al., Mol. Ther. 2011, 19:1688-1694, herein incorporated byreference in its entirety).

Liposomes, Lipoplexes, and Lipid Nanoparticles

The oncology-related polynucleotide, primary construct, and mmRNA of theinvention can be formulated using one or more liposomes, lipoplexes, orlipid nanoparticles. In one embodiment, pharmaceutical compositions ofpolynucleotide, primary construct, or mmRNA include liposomes. Liposomesare artificially-prepared vesicles which may primarily be composed of alipid bilayer and may be used as a delivery vehicle for theadministration of nutrients and pharmaceutical formulations. Liposomescan be of different sizes such as, but not limited to, a multilamellarvesicle (MLV) which may be hundreds of nanometers in diameter and maycontain a series of concentric bilayers separated by narrow aqueouscompartments, a small unicellular vesicle (SUV) which may be smallerthan 50 nm in diameter, and a large unilamellar vesicle (LUV) which maybe between 50 and 500 nm in diameter. Liposome design may include, butis not limited to, opsonins or ligands in order to improve theattachment of liposomes to unhealthy tissue or to activate events suchas, but not limited to, endocytosis. Liposomes may contain a low or ahigh pH in order to improve the delivery of the pharmaceuticalformulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;all of which are incorporated herein in their entireties). The originalmanufacture method by Wheeler et al. was a detergent dialysis method,which was later improved by Jeffs et al. and is referred to as thespontaneous vesicle formation method. The liposome formulations arecomposed of 3 to 4 lipid components in addition to the oncology-relatedpolynucleotide, primary construct, or mmRNA. As an example a liposomecan contain, but is not limited to, 55% cholesterol, 20%disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffset al. As another example, certain liposome formulations may contain,but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30%cationic lipid, where the cationic lipid can be1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described byHeyes et al.

In one embodiment, pharmaceutical compositions may include liposomeswhich may be formed to deliver mmRNA which may encode at least oneimmunogen. The mmRNA may be encapsulated by the liposome and/or it maybe contained in an aqueous core which may then be encapsulated by theliposome (see International Pub. Nos. WO2012031046, WO2012031043,WO2012030901 and WO2012006378; each of which is herein incorporated byreference in their entirety). In another embodiment, the mmRNA which mayencode an immunogen may be formulated in a cationic oil-in-wateremulsion where the emulsion particle comprises an oil core and acationic lipid which can interact with the mmRNA anchoring the moleculeto the emulsion particle (see International Pub. No. WO2012006380;herein incorporated by reference in its entirety). In yet anotherembodiment, the lipid formulation may include at least cationic lipid, alipid which may enhance transfection and a least one lipid whichcontains a hydrophilic head group linked to a lipid moiety(International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582; eachof which is herein incorporated by reference in their entirety). Inanother embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA encoding an immunogen may be formulated in alipid vesicle which may have crosslinks between functionalized lipidbilayers (see U.S. Pub. No. 20120177724, herein incorporated byreference in its entirety).

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA may be formulated in a lipid vesicle which mayhave crosslinks between functionalized lipid bilayers.

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA may be formulated in a liposome comprising acationic lipid. The liposome may have a molar ratio of nitrogen atoms inthe cationic lipid to the phophates in the RNA (N:P ratio) of between1:1 and 20:1 as described in International Publication No. WO2013006825,herein incorporated by reference in its entirety. In another embodiment,the liposome may have a N:P ratio of greater than 20:1 or less than 1:1.

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA may be formulated in a lipid-polycation complex.The formation of the lipid-polycation complex may be accomplished bymethods known in the art and/or as described in U.S. Pub. No.20120178702, herein incorporated by reference in its entirety. As anon-limiting example, the polycation may include a cationic peptide or apolypeptide such as, but not limited to, polylysine, polyornithineand/or polyarginine and the cationic peptides described in InternationalPub. No. WO2012013326; herein incorporated by reference in its entirety.In another embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA may be formulated in a lipid-polycation complexwhich may further include a neutral lipid such as, but not limited to,cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176; herein incorporated byreference in its entirety), the liposome formulation was composed of57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3%cholesterol, and 1.4% PEG-c-DMA. As another example, changing thecomposition of the cationic lipid could more effectively deliver siRNAto various antigen presenting cells (Basha et al. Mol Ther. 201119:2186-2200; herein incorporated by reference in its entirety).

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the LNP formulations. As anon-limiting example, LNP formulations may contain 1-5% of the lipidmolar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC andcholesterol. In another embodiment the PEG-c-DOMG may be replaced with aPEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In one embodiment, the polynucleotides, primary constructs or mmRNA maybe formulated in a lipid nanoparticle such as those described inInternational Publication No. WO2012170930, herein incorporated byreference in its entirety.

In one embodiment, the cationic lipid may be selected from, but notlimited to, a cationic lipid described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865 and WO2008103276, U.S. Pat. Nos. 7,893,302, 7,404,969 and8,283,333 and US Patent Publication No. US20100036115 and US20120202871;each of which is herein incorporated by reference in their entirety. Inanother embodiment, the cationic lipid may be selected from, but notlimited to, formula A described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365 and WO2012044638; each of whichis herein incorporated by reference in their entirety. In yet anotherembodiment, the cationic lipid may be selected from, but not limited to,formula CLI-CLXXIX of International Publication No. WO2008103276,formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formula CLI-CLXXXXII ofU.S. Pat. No. 7,404,969 and formula I-VI of US Patent Publication No.US20100036115; each of which is herein incorporated by reference intheir entirety. As a non-limiting example, the cationic lipid may beselected from (20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-16,19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine,(21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecyIcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amineand (l1E,20Z,23Z)—N,N-dimethylnonacosa-l1,20,2-trien-10-amine or apharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the lipid may be a cleavable lipid such as thosedescribed in International Publication No. WO2012170889, hereinincorporated by reference in its entirety.

In one embodiment, the cationic lipid may be synthesized by methodsknown in the art and/or as described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 andWO201021865; each of which is herein incorporated by reference in theirentirety.

In one embodiment, the LNP formulations of the oncology-relatedpolynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMGat 3% lipid molar ratio. In another embodiment, the LNP formulationspolynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMGat 1.5% lipid molar ratio.

In one embodiment, the pharmaceutical compositions of theoncology-related polynucleotides, primary constructs and/or mmRNA mayinclude at least one of the PEGylated lipids described in InternationalPublication No. 2012099755, herein incorporated by reference.

In one embodiment, the LNP formulation may contain PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000). In one embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art and at least one othercomponent. In another embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.As a non-limiting example, the LNP formulation may contain PEG-DMG 2000,DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNPformulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol ina molar ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral deliveryof self-amplifying RNA vaccines, PNAS 2012; PMID: 22908294; hereinincorporated by reference in its entirety). As another non-limitingexample, modified RNA described herein may be formulated in ananoparticle to be delivered by a parenteral route as described in U.S.Pub. No. 20120207845; herein incorporated by reference in its entirety.

In one embodiment, the LNP formulation may be formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276, each of which is herein incorporated by reference in theirentirety. As a non-limiting example, modified RNA described herein maybe encapsulated in LNP formulations as described in WO2011127255 and/orWO2008103276; each of which is herein incorporated by reference in theirentirety.

In one embodiment, LNP formulations described herein may comprise apolycationic composition. As a non-limiting example, the polycationiccomposition may be selected from formula 1-60 of US Patent PublicationNo. US20050222064; herein incorporated by reference in its entirety. Inanother embodiment, the LNP formulations comprising a polycationiccomposition may be used for the delivery of the modified RNA describedherein in vivo and/or in vitro.

In one embodiment, the LNP formulations described herein mayadditionally comprise a permeability enhancer molecule. Non-limitingpermeability enhancer molecules are described in US Patent PublicationNo. US20050222064; herein incorporated by reference in its entirety.

In one embodiment, the pharmaceutical compositions may be formulated inliposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutralDOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,siRNA delivery for ovarian cancer (Landen et al. Cancer Biology &Therapy 2006 5(12)1708-1713); herein incorporated by reference in itsentirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

The nanoparticle formulations may be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a modified nucleic acid molecule(e.g., mmRNA). As a non-limiting example, the carbohydrate carrier mayinclude, but is not limited to, an anhydride-modified phytoglycogen orglycogen-type material, phytoglycogen octenyl succinate, phytoglycogenbeta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g.,International Publication No. WO2012109121; herein incorporated byreference in its entirety).

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In one embodiment, the internal ester linkage may be located on eitherside of the saturated carbon. Non-limiting examples of reLNPs include,

In one embodiment, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; each of which is herein incorporated byreference in their entirety). The polymer may encapsulate thenanospecies or partially encapsulate the nanospecies. The immunogen maybe a recombinant protein, a modified RNA and/or a primary constructdescribed herein. In one embodiment, the lipid nanoparticle may beformulated for use in a vaccine such as, but not limited to, against apathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosal tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; each of which is herein incorporated by reference in theirentirety). The transport of nanoparticles may be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT). As a non-limitingexample, compositions which can penetrate a mucosal barrier may be madeas described in U.S. Pat. No. 8,241,670, herein incorporated byreference in its entirety.

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. The polymeric material mayadditionally be irradiated. As a non-limiting example, the polymericmaterial may be gamma irradiated (See e.g., International App. No.WO201282165, herein incorporated by reference in its entirety).Non-limiting examples of specific polymers include poly(caprolactone)(PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA),poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid)(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer (suchas, but not limited to, a branched polyether-polyamide block copolymerdescribed in International Publication No. WO2013012476, hereinincorporated by reference in its entirety), and (poly(ethyleneglycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblockcopolymer (see e.g., US Publication 20120121718 and US Publication20100003337 and U.S. Pat. No. 8,263,665; each of which is hereinincorporated by reference in their entirety). The co-polymer may be apolymer that is generally regarded as safe (GRAS) and the formation ofthe lipid nanoparticle may be in such a way that no new chemicalentities are created. For example, the lipid nanoparticle may comprisepoloxamers coating PLGA nanoparticles without forming new chemicalentities which are still able to rapidly penetrate human mucus (Yang etal. Angew. Chem. Int. Ed. 2011 50:2597-2600; herein incorporated byreference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, mmRNA, anionic proteins(e.g., bovine serum albumin), surfactants (e.g., cationic surfactantssuch as for example dimethyldioctadecylammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g.,N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol,sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosinβ4 dornase alfa, neltenexine, erdosteine) and various DNases includingrhDNase. The surface altering agent may be embedded or enmeshed in theparticle's surface or disposed (e.g., by coating, adsorption, covalentlinkage, or other process) on the surface of the lipid nanoparticle.(see e.g., US Publication 20100215580 and US Publication 20080166414;each of which is herein incorporated by reference in their entirety).

The mucus penetrating lipid nanoparticles may comprise at least onemmRNA described herein. The mmRNA may be encapsulated in the lipidnanoparticle and/or disposed on the surface of the particle. The mmRNAmay be covalently coupled to the lipid nanoparticle. Formulations ofmucus penetrating lipid nanoparticles may comprise a plurality ofnanoparticles. Further, the formulations may contain particles which mayinteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion which mayincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

In one embodiment, the oncology-related polynucleotide, primaryconstruct, or mmRNA is formulated as a lipoplex, such as, withoutlimitation, the ATUPLEX™ system, the DACC system, the DBTC system andother siRNA-lipoplex technology from Silence Therapeutics (London,United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.), andpolyethylenimine (PEI) or protamine-based targeted and non-targeteddelivery of nucleic acids acids (Aleku et al. Cancer Res. 200868:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78;Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 201023:334-344; Kaufmann et al. Microvasc Res 2010 80:286-293 Weide et al. JImmunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31:180-188;Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011J. Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005,23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6; 104:4095-4100;deFougerolles Hum Gene Ther. 2008 19:125-132; all of which areincorporated herein by reference in its entirety).

In one embodiment such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticleformulations which have been shown to bind to apolipoprotein E andpromote binding and uptake of these formulations into hepatocytes invivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein incorporated byreference in its entirety). Formulations can also be selectivelytargeted through expression of different ligands on their surface asexemplified by, but not limited by, folate, transferrin,N-acetylgalactosamine (GalNAc), and antibody targeted approaches(Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchioand Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol MembrBiol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst.2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhaoet al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther.2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA.2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353;Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., NatBiotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630;Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all of which areincorporated herein by reference in its entirety).

In one embodiment, the oncology-related polynucleotide, primaryconstruct, or mmRNA is formulated as a solid lipid nanoparticle. A solidlipid nanoparticle (SLN) may be spherical with an average diameterbetween 10 to 1000 nm. SLN possess a solid lipid core matrix that cansolubilize lipophilic molecules and may be stabilized with surfactantsand/or emulsifiers. In a further embodiment, the lipid nanoparticle maybe a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACSNano, 2008, 2 (8), pp 1696-1702; herein incorporated by reference in itsentirety).

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotide, primary construct, or mmRNA directed proteinproduction as these formulations may be able to increase celltransfection by the oncology-related polynucleotide, primary construct,or mmRNA; and/or increase the translation of encoded protein. One suchexample involves the use of lipid encapsulation to enable the effectivesystemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 200715:713-720; herein incorporated by reference in its entirety). Theliposomes, lipoplexes, or lipid nanoparticles may also be used toincrease the stability of the oncology-related polynucleotide, primaryconstruct, or mmRNA.

In one embodiment, the polynucleotides, primary constructs, and/or themmRNA of the present invention can be formulated for controlled releaseand/or targeted delivery. As used herein, “controlled release” refers toa pharmaceutical composition or compound release profile that conformsto a particular pattern of release to effect a therapeutic outcome. Inone embodiment, the polynucleotides, primary constructs or the mmRNA maybe encapsulated into a delivery agent described herein and/or known inthe art for controlled release and/or targeted delivery. As used herein,the term “encapsulate” means to enclose, surround or encase. As itrelates to the formulation of the compounds of the invention,encapsulation may be substantial, complete or partial. The term“substantially encapsulated” means that at least greater than 50, 60,70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999%of the pharmaceutical composition or compound of the invention may beenclosed, surrounded or encased within the delivery agent. “Partiallyencapsulation” means that less than 10, 10, 20, 30, 40 50 or less of thepharmaceutical composition or compound of the invention may be enclosed,surrounded or encased within the delivery agent. Advantageously,encapsulation may be determined by measuring the escape or the activityof the pharmaceutical composition or compound of the invention usingfluorescence and/or electron micrograph. For example, at least 1, 5, 10,20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 orgreater than 99.99% of the pharmaceutical composition or compound of theinvention are encapsulated in the delivery agent.

In one embodiment, the controlled release formulation may include, butis not limited to, tri-block co-polymers. As a non-limiting example, theformulation may include two different types of tri-block co-polymers(International Pub. No. WO2012131104 and WO2012131106; each of which isherein incorporated by reference in its entirety).

In another embodiment, the polynucleotides, primary constructs, or themmRNA may be encapsulated into a lipid nanoparticle or a rapidlyeliminated lipid nanoparticle and the lipid nanoparticles or a rapidlyeliminated lipid nanoparticle may then be encapsulated into a polymer,hydrogel and/or surgical sealant described herein and/or known in theart. As a non-limiting example, the polymer, hydrogel or surgicalsealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE®(Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics,San Diego Calif.), surgical sealants such as fibrinogen polymers(Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, IncDeerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International,Inc Deerfield, Ill.).

In another embodiment, the lipid nanoparticle may be encapsulated intoany polymer known in the art which may form a gel when injected into asubject. As another non-limiting example, the lipid nanoparticle may beencapsulated into a polymer matrix which may be biodegradable.

In one embodiment, the oncology-related polynucleotide, primaryconstruct, or mmRNA formulation for controlled release and/or targeteddelivery may also include at least one controlled release coating.Controlled release coatings include, but are not limited to, OPADRY®,polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone,hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethylcellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such asethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).

In one embodiment, the controlled release and/or targeted deliveryformulation may comprise at least one degradable polyester which maycontain polycationic side chains. Degradeable polyesters include, butare not limited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters may include a PEG conjugation toform a PEGylated polymer.

In one embodiment, the polynucleotides, primary constructs, and/or themmRNA of the present invention may be encapsulated in a therapeuticnanoparticle. Therapeutic nanoparticles may be formulated by methodsdescribed herein and known in the art such as, but not limited to,International Pub Nos. WO2010005740, WO2010030763, WO2010005721,WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645,US20100087337, US20100068285, US20110274759, US20100068286 andUS20120288541 and U.S. Pat. Nos. 8,206,747, 8,293,276, 8,318,208 and8,318,211 each of which is herein incorporated by reference in theirentirety. In another embodiment, therapeutic polymer nanoparticles maybe identified by the methods described in US Pub No. US20120140790,herein incorporated by reference in its entirety.

In one embodiment, the therapeutic nanoparticle may be formulated forsustained release. As used herein, “sustained release” refers to apharmaceutical composition or compound that conforms to a release rateover a specific period of time. The period of time may include, but isnot limited to, hours, days, weeks, months and years. As a non-limitingexample, the sustained release nanoparticle may comprise a polymer and atherapeutic agent such as, but not limited to, the polynucleotides,primary constructs, and mmRNA of the present invention (seeInternational Pub No. 2010075072 and US Pub No. US20100216804,US20110217377 and US20120201859, each of which is herein incorporated byreference in their entirety).

In one embodiment, the therapeutic nanoparticles may be formulated to betarget specific. As a non-limiting example, the therapeuticnanoparticles may include a corticosteroid (see International Pub. No.WO2011084518; herein incorporated by reference in its entirety). In oneembodiment, the therapeutic nanoparticles may be formulated to be cancerspecific. As a non-limiting example, the therapeutic nanoparticles maybe formulated in nanoparticles described in International Pub No.WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No.US20100069426, US20120004293 and US20100104655, each of which is hereinincorporated by reference in their entirety.

In one embodiment, the nanoparticles of the present invention maycomprise a polymeric matrix. As a non-limiting example, the nanoparticlemay comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblockcopolymer. In one embodiment, the diblock copolymer may include PEG incombination with a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof.

As a non-limiting example the therapeutic nanoparticle comprises aPLGA-PEG block copolymer (see US Pub. No. US20120004293 and U.S. Pat.No. 8,236,330, each of which is herein incorporated by reference intheir entirety). In another non-limiting example, the therapeuticnanoparticle is a stealth nanoparticle comprising a diblock copolymer ofPEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968 andInternational Publication No. WO2012166923, each of which is hereinincorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle may comprise amultiblock copolymer (See e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910;each of which is herein incorporated by reference in its entirety).

In one embodiment, the block copolymers described herein may be includedin a polyion complex comprising a non-polymeric micelle and the blockcopolymer. (See e.g., U.S. Pub. No. 20120076836; herein incorporated byreference in its entirety).

In one embodiment, the therapeutic nanoparticle may comprise at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In one embodiment, the therapeutic nanoparticles may comprise at leastone cationic polymer described herein and/or known in the art.

In one embodiment, the therapeutic nanoparticles may comprise at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters)(See e.g., U.S. Pat. No. 8,287,849; herein incorporated by reference inits entirety) and combinations thereof.

In one embodiment, the therapeutic nanoparticles may comprise at leastone degradable polyester which may contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters may include a PEG conjugation to form a PEGylated polymer.

In another embodiment, the therapeutic nanoparticle may include aconjugation of at least one targeting ligand. The targeting ligand maybe any ligand known in the art such as, but not limited to, a monoclonalantibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740; hereinincorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle may be formulated in anaqueous solution which may be used to target cancer (see InternationalPub No. WO2011084513 and US Pub No. US20110294717, each of which isherein incorporated by reference in their entirety).

In one embodiment, the polynucleotides, primary constructs, or mmRNA maybe encapsulated in, linked to and/or associated with syntheticnanocarriers. Synthetic nanocarriers include, but are not limited to,those described in International Pub. Nos. WO2010005740, WO2010030763,WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265,WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405,WO2012149411, WO2012149454 and WO2013019669 and US Pub. Nos.US20110262491, US20100104645, US20100087337 and US20120244222, each ofwhich is herein incorporated by reference in their entirety. Thesynthetic nanocarriers may be formulated using methods known in the artand/or described herein. As a non-limiting example, the syntheticnanocarriers may be formulated by the methods described in InternationalPub Nos. WO2010005740, WO2010030763 and WO201213501 and US Pub. Nos.US20110262491, US20100104645, US20100087337 and US2012024422, each ofwhich is herein incorporated by reference in their entirety. In anotherembodiment, the synthetic nanocarrier formulations may be lyophilized bymethods described in International Pub. No. WO2011072218 and U.S. Pat.No. 8,211,473; each of which is herein incorporated by reference intheir entirety.

In one embodiment, the synthetic nanocarriers may contain reactivegroups to release the oncology-related polynucleotides, primaryconstructs and/or mmRNA described herein (see International Pub. No.WO20120952552 and US Pub No. US20120171229, each of which is hereinincorporated by reference in their entirety).

In one embodiment, the synthetic nanocarriers may contain animmunostimulatory agent to enhance the immune response from delivery ofthe synthetic nanocarrier. As a non-limiting example, the syntheticnanocarrier may comprise a Th1 immunostimulatory agent which may enhancea Th1-based response of the immune system (see International Pub No.WO2010123569 and US Pub. No. US20110223201, each of which is hereinincorporated by reference in its entirety).

In one embodiment, the synthetic nanocarriers may be formulated fortargeted release. In one embodiment, the synthetic nanocarrier isformulated to release the oncology-related polynucleotides, primaryconstructs and/or mmRNA at a specified pH and/or after a desired timeinterval. As a non-limiting example, the synthetic nanoparticle may beformulated to release the oncology-related polynucleotides, primaryconstructs and/or mmRNA after 24 hours and/or at a pH of 4.5 (seeInternational Pub. Nos. WO2010138193 and WO2010138194 and US Pub Nos.US20110020388 and US20110027217, each of which is herein incorporated byreference in their entireties).

In one embodiment, the synthetic nanocarriers may be formulated forcontrolled and/or sustained release of the oncology-relatedpolynucleotides, primary constructs and/or mmRNA described herein. As anon-limiting example, the synthetic nanocarriers for sustained releasemay be formulated by methods known in the art, described herein and/oras described in International Pub No. WO2010138192 and US Pub No.20100303850, each of which is herein incorporated by reference in theirentirety.

In one embodiment, the synthetic nanocarrier may be formulated for useas a vaccine. In one embodiment, the synthetic nanocarrier mayencapsulate at least one polynucleotide, primary construct and/or mmRNAwhich encode at least one antigen. As a non-limiting example, thesynthetic nanocarrier may include at least one antigen and an excipientfor a vaccine dosage form (see International Pub No. WO2011150264 and USPub No. US20110293723, each of which is herein incorporated by referencein their entirety). As another non-limiting example, a vaccine dosageform may include at least two synthetic nanocarriers with the same ordifferent antigens and an excipient (see International Pub No.WO2011150249 and US Pub No. US20110293701, each of which is hereinincorporated by reference in their entirety). The vaccine dosage formmay be selected by methods described herein, known in the art and/ordescribed in International Pub No. WO2011150258 and US Pub No.US20120027806, each of which is herein incorporated by reference intheir entirety).

In one embodiment, the synthetic nanocarrier may comprise at least onepolynucleotide, primary construct and/or mmRNA which encodes at leastone adjuvant. As non-limiting example, the adjuvant may comprisedimethyldioctadecylammonium-bromide,dimethyldioctadecylammonium-chloride,dimethyldioctadecylammonium-phosphate ordimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or partof said apolar fraction of a total lipid extract of a mycobacterium (Seee.g, U.S. Pat. No. 8,241,610; herein incorporated by reference in itsentirety). In another embodiment, the synthetic nanocarrier may compriseat least one polynucleotide, primary construct and/or mmRNA and anadjuvant. As a non-limiting example, the synthetic nanocarriercomprising and adjuvant may be formulated by the methods described inInternational Pub No. WO2011150240 and US Pub No. US20110293700, each ofwhich is herein incorporated by reference in its entirety.

In one embodiment, the synthetic nanocarrier may encapsulate at leastone polynucleotide, primary construct and/or mmRNA which encodes apeptide, fragment or region from a virus. As a non-limiting example, thesynthetic nanocarrier may include, but is not limited to, thenanocarriers described in International Pub No. WO2012024621,WO201202629, WO2012024632 and US Pub No. US20120064110, US20120058153and US20120058154, each of which is herein incorporated by reference intheir entirety.

In one embodiment, the synthetic nanocarrier may be coupled to apolynucleotide, primary construct or mmRNA which may be able to triggera humoral and/or cytotoxic T lymphocyte (CTL) response (See e.g.,International Publication No. WO2013019669, herein incorporated byreference in its entirety).

In one embodiment, the nanoparticle may be optimized for oraladministration. The nanoparticle may comprise at least one cationicbiopolymer such as, but not limited to, chitosan or a derivativethereof. As a non-limiting example, the nanoparticle may be formulatedby the methods described in U.S. Pub. No. 20120282343; hereinincorporated by reference in its entirety.

Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

The oncology-related polynucleotide, primary construct, and mmRNA of theinvention can be formulated using natural and/or synthetic polymers.Non-limiting examples of polymers which may be used for deliveryinclude, but are not limited to, DYNAMIC POLYCONJUGATE® (ArrowheadReasearch Corp., Pasadena, Calif.) formulations from MIRUS® Bio(Madison, Wis.) and Roche Madison (Madison, Wis.), PHASERX® polymerformulations such as, without limitation, SMARTT POLYMER TECHNOLOGY™(Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical(San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals(Pasadena, Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA)polymers. RONDEL™ (RNAi/Oligonucleotide Nanoparticle Delivery) polymers(Arrowhead Research Corporation, Pasadena, Calif.) and pH responsiveco-block polymers such as, but not limited to, PHASERX® (Seattle,Wash.).

A non-limiting example of chitosan formulation includes a core ofpositively charged chitosan and an outer portion of negatively chargedsubstrate (U.S. Pub. No. 20120258176; herein incorporated by referencein its entirety). Chitosan includes, but is not limited to N-trimethylchitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan(NPCS), EDTA-chitosan, low molecular weight chitosan, chitosanderivatives, or combinations thereof.

In one embodiment, the polymers used in the present invention haveundergone processing to reduce and/or inhibit the attachment of unwantedsubstances such as, but not limited to, bacteria, to the surface of thepolymer. The polymer may be processed by methods known and/or describedin the art and/or described in International Pub. No. WO2012150467,herein incorporated by reference in its entirety.

A non-limiting example of PLGA formulations include, but are not limitedto, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolvingPLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueoussolvent and leuprolide. Once injected, the PLGA and leuprolide peptideprecipitates into the subcutaneous space).

Many of these polymer approaches have demonstrated efficacy indelivering oligonucleotides in vivo into the cell cytoplasm (reviewed indeFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated byreference in its entirety). Two polymer approaches that have yieldedrobust in vivo delivery of nucleic acids, in this case with smallinterfering RNA (siRNA), are dynamic polyconjugates andcyclodextrin-based nanoparticles. The first of these delivery approachesuses dynamic polyconjugates and has been shown in vivo in mice toeffectively deliver siRNA and silence endogenous target mRNA inhepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887; herein incorporated by reference in its entirety). Thisparticular approach is a multicomponent polymer system whose keyfeatures include a membrane-active polymer to which nucleic acid, inthis case siRNA, is covalently coupled via a disulfide bond and whereboth PEG (for charge masking) and N-acetylgalactosamine (for hepatocytetargeting) groups are linked via pH-sensitive bonds (Rozema et al., ProcNatl Acad Sci USA 2007 104:12982-12887; herein incorporated by referencein its entirety). On binding to the hepatocyte and entry into theendosome, the polymer complex disassembles in the low-pH environment,with the polymer exposing its positive charge, leading to endosomalescape and cytoplasmic release of the siRNA from the polymer. Throughreplacement of the N-acetylgalactosamine group with a mannose group, itwas shown one could alter targeting from asialoglycoproteinreceptor-expressing hepatocytes to sinusoidal endothelium and Kupffercells. Another polymer approach involves using transferrin-targetedcyclodextrin-containing polycation nanoparticles. These nanoparticleshave demonstrated targeted silencing of the EWS-FLI1 gene product intransferrin receptor-expressing Ewing's sarcoma tumor cells(Hu-Lieskovan et al., Cancer Res. 2005 65: 8984-8982; hereinincorporated by reference in its entirety) and siRNA formulated in thesenanoparticles was well tolerated in non-human primates (Heidel et al.,Proc Natl Acad Sci USA 2007 104:5715-21; herein incorporated byreference in its entirety). Both of these delivery strategiesincorporate rational approaches using both targeted delivery andendosomal escape mechanisms.

The polymer formulation can permit the sustained or delayed release ofpolynucleotide, primary construct, or mmRNA (e.g., followingintramuscular or subcutaneous injection). The altered release profilefor the oncology-related polynucleotide, primary construct, or mmRNA canresult in, for example, translation of an encoded protein over anextended period of time. The polymer formulation may also be used toincrease the stability of the oncology-related polynucleotide, primaryconstruct, or mmRNA. Biodegradable polymers have been previously used toprotect nucleic acids other than mmRNA from degradation and been shownto result in sustained release of payloads in vivo (Rozema et al., ProcNatl Acad Sci USA. 2007 104:12982-12887; Sullivan et al., Expert OpinDrug Deliv. 2010 7:1433-1446; Convertine et al., Biomacromolecules. 2010Oct. 1; Chu et al., Acc Chem Res. 2012 Jan. 13; Manganiello et al.,Biomaterials. 2012 33:2301-2309; Benoit et al., Biomacromolecules. 201112:2708-2714; Singha et al., Nucleic Acid Ther. 2011 2:133-147;deFougerolles Hum Gene Ther. 2008 19:125-132; Schaffert and Wagner, GeneTher. 2008 16:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 20118:1455-1468; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010464:1067-1070; each of which is herein incorporated by reference in itsentirety).

In one embodiment, the pharmaceutical compositions may be sustainedrelease formulations. In a further embodiment, the sustained releaseformulations may be for subcutaneous delivery. Sustained releaseformulations may include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

As a non-limiting example modified mRNA may be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradable,biocompatible polymers which are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine device; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C. PEG-based surgical sealants comprise two syntheticPEG components mixed in a delivery device which can be prepared in oneminute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE®and natural polymers are capable of in-situ gelation at the site ofadministration. They have been shown to interact with protein andpeptide therapeutic candidates through ionic interaction to provide astabilizing effect.

Polymer formulations can also be selectively targeted through expressionof different ligands as exemplified by, but not limited by, folate,transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; each of which is herein incorporated byreference in its entirety).

The modified nucleic acid, and mmRNA of the invention may be formulatedwith or in a polymeric compound. The polymer may include at least onepolymer such as, but not limited to, polyethenes, polyethylene glycol(PEG), poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer,biodegradable cationic lipopolymer, polyethyleneimine (PEI),cross-linked branched poly(alkylene imines), a polyamine derivative, amodified poloxamer, a biodegradable polymer, elastic biodegradablepolymer, biodegradable block copolymer, biodegradable random copolymer,biodegradable polyester copolymer, biodegradable polyester blockcopolymer, biodegradable polyester block random copolymer, multiblockcopolymers, linear biodegradable copolymer,poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradablecross-linked cationic multi-block copolymers, polycarbonates,polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), acrylic polymers, amine-containingpolymers, dextran polymers, dextran polymer derivatives or combinationsthereof.

As a non-limiting example, the modified nucleic acid or mmRNA of theinvention may be formulated with the polymeric compound of PEG graftedwith PLL as described in U.S. Pat. No. 6,177,274; herein incorporated byreference in its entirety. The formulation may be used for transfectingcells in vitro or for in vivo delivery of the modified nucleic acid andmmRNA. In another example, the modified nucleic acid and mmRNA may besuspended in a solution or medium with a cationic polymer, in a drypharmaceutical composition or in a solution that is capable of beingdried as described in U.S. Pub. Nos. 20090042829 and 20090042825; eachof which are herein incorporated by reference in their entireties.

As another non-limiting example the oncology-related polynucleotides,primary constructs or mmRNA of the invention may be formulated with aPLGA-PEG block copolymer (see US Pub. No. US20120004293 and U.S. Pat.No. 8,236,330, herein incorporated by reference in their entireties) orPLGA-PEG-PLGA block copolymers (See U.S. Pat. No. 6,004,573, hereinincorporated by reference in its entirety). As a non-limiting example,the oncology-related polynucleotides, primary constructs or mmRNA of theinvention may be formulated with a diblock copolymer of PEG and PLA orPEG and PLGA (see U.S. Pat. No. 8,246,968, herein incorporated byreference in its entirety).

A polyamine derivative may be used to deliver nucleic acids or to treatand/or prevent a disease or to be included in an implantable orinjectable device (U.S. Pub. No. 20100260817 herein incorporated byreference in its entirety). As a non-limiting example, a pharmaceuticalcomposition may include the modified nucleic acids and mmRNA and thepolyamine derivative described in U.S. Pub. No. 20100260817 (thecontents of which are incorporated herein by reference in its entirety.As a non-limiting example the oncology-related polynucleotides, primaryconstructs and mmRNA of the present invention may be delivered using apolyaminde polymer such as, but not limited to, a polymer comprising a1,3-dipolar addition polymer prepared by combining a carbohydratediazide monomer with a dilkyne unite comprising oligoamines (U.S. Pat.No. 8,236,280; herein incorporated by reference in its entirety).

In one embodiment, the oncology-related polynucleotides, primaryconstructs or mmRNA of the present invention may be formulated with atleast one polymer and/or derivatives thereof described in InternationalPublication Nos. WO2011115862, WO2012082574 and WO2012068187 and U.S.Pub. No. 20120283427, each of which are herein incorporated by referencein their entireties. In another embodiment, the modified nucleic acid ormmRNA of the present invention may be formulated with a polymer offormula Z as described in WO2011115862, herein incorporated by referencein its entirety. In yet another embodiment, the modified nucleic acid ormmRNA may be formulated with a polymer of formula Z, Z′ or Z″ asdescribed in International Pub. Nos. WO2012082574 or WO2012068187 andU.S. Pub. No. 2012028342, each of which are herein incorporated byreference in their entireties. The polymers formulated with the modifiedRNA of the present invention may be synthesized by the methods describedin International Pub. Nos. WO2012082574 or WO2012068187, each of whichare herein incorporated by reference in their entireties.

The oncology-related polynucleotides, primary constructs or mmRNA of theinvention may be formulated with at least one acrylic polymer. Acrylicpolymers include but are not limited to, acrylic acid, methacrylic acid,acrylic acid and methacrylic acid copolymers, methyl methacrylatecopolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylicacid), polycyanoacrylates and combinations thereof.

Formulations of oncology-related polynucleotides, primary constructs ormmRNA of the invention may include at least one amine-containing polymersuch as, but not limited to polylysine, polyethylene imine,poly(amidoamine) dendrimers or combinations thereof.

For example, the modified nucleic acid or mmRNA of the invention may beformulated in a pharmaceutical compound including a poly(alkyleneimine), a biodegradable cationic lipopolymer, a biodegradable blockcopolymer, a biodegradable polymer, or a biodegradable random copolymer,a biodegradable polyester block copolymer, a biodegradable polyesterpolymer, a biodegradable polyester random copolymer, a linearbiodegradable copolymer, PAGA, a biodegradable cross-linked cationicmulti-block copolymer or combinations thereof. The biodegradablecationic lipopolymer may be made by methods known in the art and/ordescribed in U.S. Pat. No. 6,696,038, U.S. App. Nos. 20030073619 and20040142474 each of which is herein incorporated by reference in theirentireties. The poly(alkylene imine) may be made using methods known inthe art and/or as described in U.S. Pub. No. 20100004315, hereinincorporated by reference in its entirety. The biodegradable polymer,biodegradable block copolymer, the biodegradable random copolymer,biodegradable polyester block copolymer, biodegradable polyesterpolymer, or biodegradable polyester random copolymer may be made usingmethods known in the art and/or as described in U.S. Pat. Nos. 6,517,869and 6,267,987, the contents of which are each incorporated herein byreference in their entirety. The linear biodegradable copolymer may bemade using methods known in the art and/or as described in U.S. Pat. No.6,652,886. The PAGA polymer may be made using methods known in the artand/or as described in U.S. Pat. No. 6,217,912 herein incorporated byreference in its entirety. The PAGA polymer may be copolymerized to forma copolymer or block copolymer with polymers such as but not limited to,poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines,polylactides and poly(lactide-co-glycolides). The biodegradablecross-linked cationic multi-block copolymers may be made my methodsknown in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S.Pub. No. 2012009145 each of which are herein incorporated by referencein their entireties. For example, the multi-block copolymers may besynthesized using linear polyethyleneimine (LPEI) blocks which havedistinct patterns as compared to branched polyethyleneimines. Further,the composition or pharmaceutical composition may be made by the methodsknown in the art, described herein, or as described in U.S. Pub. No.20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912 each of which areherein incorporated by reference in their entireties.

The polynucleotides, primary constructs, and mmRNA of the invention maybe formulated with at least one degradable polyester which may containpolycationic side chains. Degradeable polyesters include, but are notlimited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters may include a PEG conjugation toform a PEGylated polymer.

The polynucleotides, primary construct, mmRNA of the invention may beformulated with at least one crosslinkable polyester. Crosslinkablepolyesters include those known in the art and described in US Pub. No.20120269761, herein incorporated by reference in its entirety.

In one embodiment, the polymers described herein may be conjugated to alipid-terminating PEG. As a non-limiting example, PLGA may be conjugatedto a lipid-terminating PEG forming PLGA-DSPE-PEG. As anothernon-limiting example, PEG conjugates for use with the present inventionare described in International Publication No. WO2008103276, hereinincorporated by reference in its entirety. The polymers may beconjugated using a ligand conjugate such as, but not limited to, theconjugates described in U.S. Pat. No. 8,273,363, herein incorporated byreference in its entirety.

In one embodiment, the modified RNA described herein may be conjugatedwith another compound. Non-limiting examples of conjugates are describedin U.S. Pat. Nos. 7,964,578 and 7,833,992, each of which are hereinincorporated by reference in their entireties. In another embodiment,modified RNA of the present invention may be conjugated with conjugatesof formula 1-122 as described in U.S. Pat. Nos. 7,964,578 and 7,833,992,each of which are herein incorporated by reference in their entireties.The oncology-related polynucleotides, primary constructs and/or mmRNAdescribed herein may be conjugated with a metal such as, but not limitedto, gold. (See e.g., Giljohann et al. Journ. Amer. Chem. Soc. 2009131(6): 2072-2073; herein incorporated by reference in its entirety). Inanother embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA described herein may be conjugated and/orencapsulated in gold-nanoparticles. (Interantional Pub. No. WO201216269and U.S. Pub. No. 20120302940; each of which is herein incorporated byreference in its entirety).

As described in U.S. Pub. No. 20100004313, herein incorporated byreference in its entirety, a gene delivery composition may include anucleotide sequence and a poloxamer. For example, the modified nucleicacid and mmRNA of the present invention may be used in a gene deliverycomposition with the poloxamer described in U.S. Pub. No. 20100004313.

In one embodiment, the polymer formulation of the present invention maybe stabilized by contacting the polymer formulation, which may include acationic carrier, with a cationic lipopolymer which may be covalentlylinked to cholesterol and polyethylene glycol groups. The polymerformulation may be contacted with a cationic lipopolymer using themethods described in U.S. Pub. No. 20090042829 herein incorporated byreference in its entirety. The cationic carrier may include, but is notlimited to, polyethylenimine, poly(trimethylenimine),poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine,dideoxy-diamino-b-cyclodextrin, spermine, spermidine,poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine),poly(arginine), cationized gelatin, dendrimers, chitosan,1,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),3B—[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl) diheptadecylamidoglycyl spermidine (DOGS),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) andcombinations thereof.

The oncology-related polynucleotides, primary constructs and/or mmRNA ofthe invention may be formulated in a polyplex of one or more polymers(U.S. Pub. No. 20120237565 and 20120270927; each of which is hereinincorporated by reference in its entirety). In one embodiment, thepolyplex comprises two or more cationic polymers. The catioinic polymermay comprise a poly(ethylene imine) (PEI) such as linear PEI.

The oncology-related polynucleotide, primary construct, and mmRNA of theinvention can also be formulated as a nanoparticle using a combinationof polymers, lipids, and/or other biodegradable agents, such as, but notlimited to, calcium phosphate. Components may be combined in acore-shell, hybrid, and/or layer-by-layer architecture, to allow forfine-tuning of the nanoparticle so to delivery of the polynucleotide,primary construct and mmRNA may be enhanced (Wang et al., Nat Mater.2006 5:791-796; Fuller et al., Biomaterials. 2008 29:1526-1532; DeKokeret al., Adv Drug Deliv Rev. 2011 63:748-761; Endres et al.,Biomaterials. 2011 32:7721-7731; Su et al., Mol Pharm. 2011 Jun. 6;8(3):774-87; herein incorporated by reference in its entirety). As anon-limiting example, the nanoparticle may comprise a plurality ofpolymers such as, but not limited to hydrophilic-hydrophobic polymers(e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilicpolymers (International Pub. No. WO20120225129; herein incorporated byreference in its entirety).

Biodegradable calcium phosphate nanoparticles in combination with lipidsand/or polymers have been shown to deliver polynucleotides, primaryconstructs and mmRNA in vivo. In one embodiment, a lipid coated calciumphosphate nanoparticle, which may also contain a targeting ligand suchas anisamide, may be used to deliver the polynucleotide, primaryconstruct and mmRNA of the present invention. For example, toeffectively deliver siRNA in a mouse metastatic lung model a lipidcoated calcium phosphate nanoparticle was used (Li et al., J Contr Rel.2010 142: 416-421; Li et al., J Contr Rel. 2012 158:108-114; Yang etal., Mol Ther. 2012 20:609-615; herein incorporated by reference in itsentirety). This delivery system combines both a targeted nanoparticleand a component to enhance the endosomal escape, calcium phosphate, inorder to improve delivery of the siRNA.

In one embodiment, calcium phosphate with a PEG-polyanion blockcopolymer may be used to delivery polynucleotides, primary constructsand mmRNA (Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa etal., J Contr Rel. 2006 111:368-370; herein incorporated by reference inits entirety).

In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle todeliver the oncology-related polynucleotides, primary constructs andmmRNA of the present invention. The PEG-charge-conversional polymer mayimprove upon the PEG-polyanion block copolymers by being cleaved into apolycation at acidic pH, thus enhancing endosomal escape.

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001). The complexation, delivery, and internalization of thepolymeric nanoparticles can be precisely controlled by altering thechemical composition in both the core and shell components of thenanoparticle. For example, the core-shell nanoparticles may efficientlydeliver siRNA to mouse hepatocytes after they covalently attachcholesterol to the nanoparticle.

In one embodiment, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer containing PEG may be used to deliveryof the polynucleotide, primary construct and mmRNA of the presentinvention. As a non-limiting example, in mice bearing aluciferase-expressing tumor, it was determined that thelipid-polymer-lipid hybrid nanoparticle significantly suppressedluciferase expression, as compared to a conventional lipoplex (Shi etal, Angew Chem Int Ed. 2011 50:7027-7031; herein incorporated byreference in its entirety).

In one embodiment, the lipid nanoparticles may comprise a core of themodified nucleic acid molecules disclosed herein and a polymer shell.The polymer shell may be any of the polymers described herein and areknown in the art. In an additional embodiment, the polymer shell may beused to protect the modified nucleic acids in the core.

Core-shell nanoparticles for use with the modified nucleic acidmolecules of the present invention are described and may be formed bythe methods described in U.S. Pat. No. 8,313,777 herein incorporated byreference in its entirety.

In one embodiment, the core-shell nanoparticles may comprise a core ofthe modified nucleic acid molecules disclosed herein and a polymershell. The polymer shell may be any of the polymers described herein andare known in the art. In an additional embodiment, the polymer shell maybe used to protect the modified nucleic acid molecules in the core. As anon-limiting example, the core-shell nanoparticle may be used to treatan eye disease or disorder (See e.g. US Publication No. 20120321719,herein incorporated by reference in its entirety).

In one embodiment, the polymer used with the formulations describedherein may be a modified polymer (such as, but not limited to, amodified polyacetal) as described in International Publication No.WO2011120053, herein incorporated by reference in its entirety.

Peptides and Proteins

The oncology-related polynucleotide, primary construct, and mmRNA of theinvention can be formulated with peptides and/or proteins in order toincrease transfection of cells by the oncology-related polynucleotide,primary construct, or mmRNA. In one embodiment, peptides such as, butnot limited to, cell penetrating peptides and proteins and peptides thatenable intracellular delivery may be used to deliver pharmaceuticalformulations. A non-limiting example of a cell penetrating peptide whichmay be used with the pharmaceutical formulations of the presentinvention includes a cell-penetrating peptide sequence attached topolycations that facilitates delivery to the intracellular space, e.g.,HIV-derived TAT peptide, penetratins, transportans, or hCT derivedcell-penetrating peptides (see, e.g., Caron et al., Mol. Ther.3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes andApplications (CRC Press, Boca Raton Fla., 2002); El-Andaloussi et al.,Curr. Pharm. Des. 11(28):3597-611 (2003); and Deshayes et al., Cell.Mol. Life Sci. 62(16):1839-49 (2005), all of which are incorporatedherein by reference in their entirety). The compositions can also beformulated to include a cell penetrating agent, e.g., liposomes, whichenhance delivery of the compositions to the intracellular space.Polynucleotides, primary constructs, and mmRNA of the invention may becomplexed to peptides and/or proteins such as, but not limited to,peptides and/or proteins from Aileron Therapeutics (Cambridge, Mass.)and Permeon Biologics (Cambridge, Mass.) in order to enableintracellular delivery (Cronican et al., ACS Chem. Biol. 2010 5:747-752;McNaughton et al., Proc. Natl. Acad. Sci. USA 2009 106:6111-6116;Sawyer, Chem Biol Drug Des. 2009 73:3-6; Verdine and Hilinski, MethodsEnzymol. 2012; 503:3-33; all of which are herein incorporated byreference in its entirety).

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but are not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the oncology-related polynucleotide, primary construct, ormmRNA may be introduced.

Formulations of the including peptides or proteins may be used toincrease cell transfection by the oncology-related polynucleotide,primary construct, or mmRNA, alter the biodistribution of theoncology-related polynucleotide, primary construct, or mmRNA (e.g., bytargeting specific tissues or cell types), and/or increase thetranslation of encoded protein. (See e.g., International Pub. No.WO2012110636; herein incorporated by reference in its entirety).

Cells

The oncology-related polynucleotide, primary construct, and mmRNA of theinvention can be transfected ex vivo into cells, which are subsequentlytransplanted into a subject. As non-limiting examples, thepharmaceutical compositions may include red blood cells to delivermodified RNA to liver and myeloid cells, virosomes to deliver modifiedRNA in virus-like particles (VLPs), and electroporated cells such as,but not limited to, from MAXCYTE® (Gaithersburg, Md.) and from ERYTECH®(Lyon, France) to deliver modified RNA. Examples of use of red bloodcells, viral particles and electroporated cells to deliver payloadsother than mmRNA have been documented (Godfrin et al., Expert Opin BiolTher. 2012 12:127-133; Fang et al., Expert Opin Biol Ther. 201212:385-389; Hu et al., Proc Natl Acad Sci USA. 2011 108:10980-10985;Lund et al., Pharm Res. 2010 27:400-420; Huckriede et al., J LiposomeRes. 2007; 17:39-47; Cusi, Hum Vaccin. 2006 2:1-7; de Jonge et al., GeneTher. 2006 13:400-411; all of which are herein incorporated by referencein its entirety).

The oncology-related polynucleotides, primary constructs and mmRNA maybe delivered in synthetic VLPs synthesized by the methods described inInternational Pub No. WO2011085231 and US Pub No. 20110171248, each ofwhich are herein incorporated by reference in their entireties.

Cell-based formulations of the oncology-related polynucleotide, primaryconstruct, and mmRNA of the invention may be used to ensure celltransfection (e.g., in the cellular carrier), alter the biodistributionof the oncology-related polynucleotide, primary construct, or mmRNA(e.g., by targeting the cell carrier to specific tissues or cell types),and/or increase the translation of encoded protein.

A variety of methods are known in the art and suitable for introductionof nucleic acid into a cell, including viral and non-viral mediatedtechniques. Examples of typical non-viral mediated techniques include,but are not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microproj ectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion.

The technique of sonoporation, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to those in theart and are used to deliver nucleic acids in vivo (Yoon and Park, ExpertOpin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr PharmBiotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 200714:465-475; all herein incorporated by reference in their entirety).Sonoporation methods are known in the art and are also taught forexample as it relates to bacteria in US Patent Publication 20100196983and as it relates to other cell types in, for example, US PatentPublication 20100009424, each of which are incorporated herein byreference in their entirety.

Electroporation techniques are also well known in the art and are usedto deliver nucleic acids in vivo and clinically (Andre et al., Curr GeneTher. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 201010:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all hereinincorporated by reference in their entirety). In one embodiment,polynucleotides, primary constructs or mmRNA may be delivered byelectroporation as described in Example 8.

Hyaluronidase

The intramuscular or subcutaneous localized injection of polynucleotide,primary construct, or mmRNA of the invention can include hyaluronidase,which catalyzes the hydrolysis of hyaluronan. By catalyzing thehydrolysis of hyaluronan, a constituent of the interstitial barrier,hyaluronidase lowers the viscosity of hyaluronan, thereby increasingtissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440;herein incorporated by reference in its entirety). It is useful to speedtheir dispersion and systemic distribution of encoded proteins producedby transfected cells. Alternatively, the hyaluronidase can be used toincrease the number of cells exposed to a oncology-relatedpolynucleotide, primary construct, or mmRNA of the inventionadministered intramuscularly or subcutaneously.

Nanoparticle Mimics

The oncology-related polynucleotide, primary construct or mmRNA of theinvention may be encapsulated within and/or absorbed to a nanoparticlemimic. A nanoparticle mimic can mimic the delivery function organisms orparticles such as, but not limited to, pathogens, viruses, bacteria,fungus, parasites, prions and cells. As a non-limiting example theoncology-related polynucleotide, primary construct or mmRNA of theinvention may be encapsulated in a non-viron particle which can mimicthe delivery function of a virus (see International Pub. No.WO2012006376 herein incorporated by reference in its entirety).

Nanotubes

The oncology-related polynucleotides, primary constructs or mmRNA of theinvention can be attached or otherwise bound to at least one nanotubesuch as, but not limited to, rosette nanotubes, rosette nanotubes havingtwin bases with a linker, carbon nanotubes and/or single-walled carbonnanotubes, The oncology-related polynucleotides, primary constructs ormmRNA may be bound to the nanotubes through forces such as, but notlimited to, steric, ionic, covalent and/or other forces.

In one embodiment, the nanotube can release one or more polynucleotides,primary constructs or mmRNA into cells. The size and/or the surfacestructure of at least one nanotube may be altered so as to govern theinteraction of the nanotubes within the body and/or to attach or bind tothe oncology-related polynucleotides, primary constructs or mmRNAdisclosed herein. In one embodiment, the building block and/or thefunctional groups attached to the building block of the at least onenanotube may be altered to adjust the dimensions and/or properties ofthe nanotube. As a non-limiting example, the length of the nanotubes maybe altered to hinder the nanotubes from passing through the holes in thewalls of normal blood vessels but still small enough to pass through thelarger holes in the blood vessels of tumor tissue.

In one embodiment, at least one nanotube may also be coated withdelivery enhancing compounds including polymers, such as, but notlimited to, polyethylene glycol. In another embodiment, at least onenanotube and/or the oncology-related polynucleotides, primary constructsor mmRNA may be mixed with pharmaceutically acceptable excipients and/ordelivery vehicles.

In one embodiment, the oncology-related polynucleotides, primaryconstructs or mmRNA are attached and/or otherwise bound to at least onerosette nanotube. The rosette nanotubes may be formed by a process knownin the art and/or by the process described in International PublicationNo. WO2012094304, herein incorporated by reference in its entirety. Atleast one polynucleotide, primary construct and/or mmRNA may be attachedand/or otherwise bound to at least one rosette nanotube by a process asdescribed in International Publication No. WO2012094304, hereinincorporated by reference in its entirety, where rosette nanotubes ormodules forming rosette nanotubes are mixed in aqueous media with atleast one polynucleotide, primary construct and/or mmRNA underconditions which may cause at least one polynucleotide, primaryconstruct or mmRNA to attach or otherwise bind to the rosette nanotubes.

In one embodiment, the oncology-related polynucleotides, primaryconstructs or mmRNA may be attached to and/or otherwise bound to atleast one carbon nanotube. As a non-limiting example, theoncology-related polynucleotides, primary constructs or mmRNA may bebound to a linking agent and the linked agent may be bound to the carbonnanotube (See e.g., U.S. Pat. No. 8,246,995; herein incorporated byreference in its entirety). The carbon nanotube may be a single-wallednanotube (See e.g., U.S. Pat. No. 8,246,995; herein incorporated byreference in its entirety).

Conjugates

The polynucleotides, primary constructs, and mmRNA of the inventioninclude conjugates, such as a oncology-related polynucleotide, primaryconstruct, or mmRNA covalently linked to a carrier or targeting group,or including two encoding regions that together produce a fusion protein(e.g., bearing a targeting group and therapeutic protein or peptide).

The conjugates of the invention include a naturally occurring substance,such as a protein (e.g., human serum albumin (HSA), low-densitylipoprotein (LDL), high-density lipoprotein (HDL), or globulin); ancarbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin,cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examplesof polyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Representative U.S. patents that teach the preparation of polynucleotideconjugates, particularly to RNA, include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664;6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which isherein incorporated by reference in their entireties.

In one embodiment, the conjugate of the present invention may functionas a carrier for the modified nucleic acids and mmRNA of the presentinvention. The conjugate may comprise a cationic polymer such as, butnot limited to, polyamine, polylysine, polyalkylenimine, andpolyethylenimine which may be grafted to with poly(ethylene glycol). Asa non-limiting example, the conjugate may be similar to the polymericconjugate and the method of synthesizing the polymeric conjugatedescribed in U.S. Pat. No. 6,586,524 herein incorporated by reference inits entirety.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell. Targeting groups mayalso include hormones and hormone receptors. They can also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose,multivalent fucose, or aptamers. The ligand can be, for example, alipopolysaccharide, or an activator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GalNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein.

In one embodiment, pharmaceutical compositions of the present inventionmay include chemical modifications such as, but not limited to,modifications similar to locked nucleic acids.

Representative U.S. patents that teach the preparation of locked nucleicacid (LNA) such as those from Santaris, include, but are not limited to,the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is hereinincorporated by reference in its entirety.

Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found, for example, in Nielsenet al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include polynucleotides,primary constructs or mmRNA with phosphorothioate backbones andoligonucleosides with other modified backbones, and in particular—CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) orMMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone isrepresented as —O—P(O)₂—O—CH₂—] of the above-referenced U.S. Pat. No.5,489,677, and the amide backbones of the above-referenced U.S. Pat. No.5,602,240. In some embodiments, the polynucleotides featured herein havemorpholino backbone structures of the above-referenced U.S. Pat. No.5,034,506.

Modifications at the 2′ position may also aid in delivery. Preferably,modifications at the 2′ position are not located in a polypeptide-codingsequence, i.e., not in a translatable region. Modifications at the 2′position may be located in a 5′UTR, a 3′UTR and/or a tailing region.Modifications at the 2′ position can include one of the following at the2′ position: H (i.e., 2′-deoxy); F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, the oncology-relatedpolynucleotides, primary constructs or mmRNA include one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties, or a group for improving thepharmacodynamic properties, and other substituents having similarproperties. In some embodiments, the modification includes a2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., analkoxy-alkoxy group. Another exemplary modification is2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)2, also described in examples herein below. Othermodifications include 2′-methoxy(2′-OCH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. Polynucleotides ofthe invention may also have sugar mimetics such as cyclobutyl moietiesin place of the pentofuranosyl sugar. Representative U.S. patents thatteach the preparation of such modified sugar structures include, but arenot limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920 and each ofwhich is herein incorporated by reference.

In still other embodiments, the oncology-related polynucleotide, primaryconstruct, or mmRNA is covalently conjugated to a cell penetratingpolypeptide. The cell-penetrating peptide may also include a signalsequence. The conjugates of the invention can be designed to haveincreased stability; increased cell transfection; and/or altered thebiodistribution (e.g., targeted to specific tissues or cell types).

In one embodiment, the polynucleotides, primary constructs or mmRNA maybe conjugated to an agent to enhance delivery. As a non-limitingexample, the agent may be a monomer or polymer such as a targetingmonomer or a polymer having targeting blocks as described inInternational Publication No. WO2011062965, herein incorporated byreference in its entirety. In another non-limiting example, the agentmay be a transport agent covalently coupled to the polynucleotides,primary constructs or mmRNA of the present invention (See e.g., U.S.Pat. Nos. 6,835,393 and 7,374,778, each of which is herein incorporatedby reference in its entirety). In yet another non-limiting example, theagent may be a membrane barrier transport enhancing agent such as thosedescribed in U.S. Pat. Nos. 7,737,108 and 8,003,129, each of which isherein incorporated by reference in its entirety.

In another embodiment, polynucleotides, primary constructs or mmRNA maybe conjugated to SMARTT POLYMER TECHNOLOGY® (PHASERX®, Inc. Seattle,Wash.).

Self-Assembled Nanoparticles Nucleic Acid Self-Assembled Nanoparticles

Self-assembled nanoparticles have a well-defined size which may beprecisely controlled as the nucleic acid strands may be easilyreprogrammable. For example, the optimal particle size for acancer-targeting nanodelivery carrier is 20-100 nm as a diameter greaterthan 20 nm avoids renal clearance and enhances delivery to certaintumors through enhanced permeability and retention effect. Usingself-assembled nucleic acid nanoparticles a single uniform population insize and shape having a precisely controlled spatial orientation anddensity of cancer-targeting ligands for enhanced delivery. As anon-limiting example, oligonucleotide nanoparticles were prepared usingprogrammable self-assembly of short DNA fragments and therapeuticsiRNAs. These nanoparticles are molecularly identical with controllableparticle size and target ligand location and density. The DNA fragmentsand siRNAs self-assembled into a one-step reaction to generate DNA/siRNAtetrahedral nanoparticles for targeted in vivo delivery. (Lee et al.,Nature Nanotechnology 2012 7:389-393; herein incorporated by referencein its entirety).

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA disclosed herein may be formulated asself-assembled nanoparticles. As a non-limiting example, nucleic acidsmay be used to make nanoparticles which may be used in a delivery systemfor the oncology-related polynucleotides, primary constructs and/ormmRNA of the present invention (See e.g., International Pub. No.WO2012125987; herein incorporated by reference in its entirety).

In one embodiment, the nucleic acid self-assembled nanoparticles maycomprise a core of the oncology-related polynucleotides, primaryconstructs or mmRNA disclosed herein and a polymer shell. The polymershell may be any of the polymers described herein and are known in theart. In an additional embodiment, the polymer shell may be used toprotect the polynucleotides, primary contructs and mmRNA in the core.

Polymer-Based Self-Assembled Nanoparticles

Polymers may be used to form sheets which self-assembled intonanoparticles. These nanoparticles may be used to deliver theoncology-related polynucleotides, primary constructs and mmRNA of thepresent invention. In one embodiment, these self-assembled nanoparticlesmay be microsponges formed of long polymers of RNA hairpins which forminto crystalline ‘pleated’ sheets before self-assembling intomicrosponges. These microsponges are densely-packed sponge likemicroparticles which may function as an efficient carrier and may beable to deliver cargo to a cell. The microsponges may be from 1 um to300 nm in diameter. The microsponges may be complexed with other agentsknown in the art to form larger microsponges. As a non-limiting example,the microsponge may be complexed with an agent to form an outer layer topromote cellular uptake such as polycation polyethyleneime (PEI). Thiscomplex can form a 250-nm diameter particle that can remain stable athigh temperatures (150° C.) (Grabow and Jaegar, Nature Materials 2012,11:269-269; herein incorporated by reference in its entirety).Additionally these microsponges may be able to exhibit an extraordinarydegree of protection from degradation by ribonucleases.

In another embodiment, the polymer-based self-assembled nanoparticlessuch as, but not limited to, microsponges, may be fully programmablenanoparticles. The geometry, size and stoichiometry of the nanoparticlemay be precisely controlled to create the optimal nanoparticle fordelivery of cargo such as, but not limited to, polynucleotides, primaryconstructs and/or mmRNA.

In one embodiment, the polymer based nanoparticles may comprise a coreof the oncology-related polynucleotides, primary constructs and/or mmRNAdisclosed herein and a polymer shell. The polymer shell may be any ofthe polymers described herein and are known in the art. In an additionalembodiment, the polymer shell may be used to protect thepolynucleotides, primary construct and/or mmRNA in the core.

In yet another embodiment, the polymer based nanoparticle may comprise anon-nucleic acid polymer comprising a plurality of heterogenous monomerssuch as those described in Interantional Publication No. WO2013009736,herein incorporated by reference in its entirety.

Inorganic Nanoparticles

The oncology-related polynucleotides, primary constructs and/or mmRNAsof the present invention may be formulated in inorganic nanoparticles(U.S. Pat. No. 8,257,745, herein incorporated by reference in itsentirety). The inorganic nanoparticles may include, but are not limitedto, clay substances that are water swellable. As a non-limiting example,the inorganic nanoparticle may include synthetic smectite clays whichare made from simple silicates (See e.g., U.S. Pat. Nos. 5,585,108 and8,257,745 each of which are herein incorporated by reference in theirentirety).

In one embodiment, the inorganic nanoparticles may comprise a core ofthe modified nucleic acids disclosed herein and a polymer shell. Thepolymer shell may be any of the polymers described herein and are knownin the art. In an additional embodiment, the polymer shell may be usedto protect the modified nucleic acids in the core.

Semi-Conductive and Metallic Nanoparticles

The oncology-related polynucleotides, primary constructs and/or mmRNAsof the present invention may be formulated in water-dispersiblenanoparticle comprising a semiconductive or metallic material (U.S. Pub.No. 20120228565; herein incorporated by reference in its entirety) orformed in a magnetic nanoparticle (U.S. Pub. No. 20120265001 and20120283503; each of which is herein incorporated by reference in itsentirety). The water-dispersible nanoparticles may be hydrophobicnanoparticles or hydrophilic nanoparticles.

In one embodiment, the semi-conductive and/or metallic nanoparticles maycomprise a core of the oncology-related polynucleotides, primaryconstructs and/or mmRNA disclosed herein and a polymer shell. Thepolymer shell may be any of the polymers described herein and are knownin the art. In an additional embodiment, the polymer shell may be usedto protect the oncology-related polynucleotides, primary constructsand/or mmRNA in the core.

Gels and Hydrogels

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA disclosed herein may be encapsulated into anyhydrogel known in the art which may form a gel when injected into asubject. Hydrogels are a network of polymer chains that are hydrophilic,and are sometimes found as a colloidal gel in which water is thedispersion medium. Hydrogels are highly absorbent (they can contain over99% water) natural or synthetic polymers. Hydrogels also possess adegree of flexibility very similar to natural tissue, due to theirsignificant water content. The hydrogel described herein may used toencapsulate lipid nanoparticles which are biocompatible, biodegradableand/or porous.

As a non-limiting example, the hydrogel may be an aptamer-functionalizedhydrogel. The aptamer-functionalized hydrogel may be programmed torelease one or more polynucleotides, primary constructs and/or mmRNAusing nucleic acid hybridization. (Battig et al., J. Am. Chem. Society.2012 134:12410-12413; herein incorporated by reference in its entirety).

As another non-limiting example, the hydrogel may be a shaped as aninverted opal.

The opal hydrogels exhibit higher swelling ratios and the swellingkinetics is an order of magnitude faster as well. Methods of producingopal hydrogels and description of opal hydrogels are described inInternational Pub. No. WO2012148684, herein incorporated by reference inits entirety.

In yet another non-limiting example, the hydrogel may be anantibacterial hydrogel. The antibacterial hydrogel may comprise apharmaceutical acceptable salt or organic material such as, but notlimited to pharmaceutical grade and/or medical grade silver salt andaloe vera gel or extract. (International Pub. No. WO2012151438, hereinincorporated by reference in its entirety).

In one embodiment, the modified mRNA may be encapsulated in a lipidnanoparticle and then the lipid nanoparticle may be encapsulated into ahyrdogel.

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA disclosed herein may be encapsulated into anygel known in the art. As a non-limiting example the gel may be afluorouracil injectable gel or a fluorouracil injectable gel containinga chemical compound and/or drug known in the art. As another example,the oncology-related polynucleotides, primary constructs and/or mmRNAmay be encapsulated in a fluorouracil gel containing epinephrine (Seee.g., Smith et al. Cancer Chemotherapty and Pharmacology, 199944(4):267-274; herein incorporated by reference in its entirety).

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA disclosed herein may be encapsulated into afibrin gel, fibrin hydrogel or fibrin glue. In another embodiment, theoncology-related polynucleotides, primary constructs and/or mmRNA may beformulated in a lipid nanoparticle or a rapidly eliminated lipidnanoparticle prior to being encapsulated into a fibrin gel, fibrinhydrogel or a fibrin glue. In yet another embodiment, theoncology-related polynucleotides, primary constructs and/or mmRNA may beformulated as a lipoplex prior to being encapsulated into a fibrin gel,hydrogel or a fibrin glue. Fibrin gels, hydrogels and glues comprise twocomponents, a fibrinogen solution and a thrombin solution which is richin calcium (See e.g., Spicer and Mikos, Journal of Controlled Release2010. 148: 49-55; Kidd et al. Journal of Controlled Release 2012.157:80-85; each of which is herein incorporated by reference in itsentirety). The concentration of the components of the fibrin gel,hydrogel and/or glue can be altered to change the characteristics, thenetwork mesh size, and/or the degradation characteristics of the gel,hydrogel and/or glue such as, but not limited to changing the releasecharacteristics of the fibrin gel, hydrogel and/or glue. (See e.g.,Spicer and Mikos, Journal of Controlled Release 2010. 148: 49-55; Kiddet al. Journal of Controlled Release 2012. 157:80-85; Catelas et al.Tissue Engineering 2008. 14:119-128; each of which is hereinincorporated by reference in its entirety). This feature may beadvantageous when used to deliver the modified mRNA disclosed herein.(See e.g., Kidd et al. Journal of Controlled Release 2012. 157:80-85;Catelas et al. Tissue Engineering 2008. 14:119-128; each of which isherein incorporated by reference in its entirety).

Cations and Anions

Formulations of polynucleotides, primary constructs and/or mmRNAdisclosed herein may include cations or anions. In one embodiment, theformulations include metal cations such as, but not limited to, Zn2+,Ca2+, Cu2+, Mg+ and combinations thereof. As a non-limiting example,formulations may include polymers and a polynucleotides, primaryconstructs and/or mmRNA complexed with a metal cation (See e.g., U.S.Pat. Nos. 6,265,389 and 6,555,525, each of which is herein incorporatedby reference in its entirety).

Molded Nanoparticles and Microparticles

The oncology-related polynucleotides, primary constructs and/or mmRNAdisclosed herein may be formulated in nanoparticles and/ormicroparticles. These nanoparticles and/or microparticles may be moldedinto any size shape and chemistry. As an example, the nanoparticlesand/or microparticles may be made using the PRINT® technology by LIQUIDATECHNOLOGIES® (Morrisville, N.C.) (See e.g., International Pub. No.WO2007024323; herein incorporated by reference in its entirety).

In one embodiment, the molded nanoparticles may comprise a core of theoncology-related polynucleotides, primary constructs and/or mmRNAdisclosed herein and a polymer shell. The polymer shell may be any ofthe polymers described herein and are known in the art. In an additionalembodiment, the polymer shell may be used to protect thepolynucleotides, primary construct and/or mmRNA in the core.

NanoJackets and NanoLiposomes

The oncology-related polynucleotides, primary constructs and/or mmRNAdisclosed herein may be formulated in NanoJackets and NanoLiposomes byKeystone Nano (State College, Pa.). NanoJackets are made of compoundsthat are naturally found in the body including calcium, phosphate andmay also include a small amount of silicates. Nanojackets may range insize from 5 to 50 nm and may be used to deliver hydrophilic andhydrophobic compounds such as, but not limited to, polynucleotides,primary constructs and/or mmRNA.

NanoLiposomes are made of lipids such as, but not limited to, lipidswhich naturally occur in the body. NanoLiposomes may range in size from60-80 nm and may be used to deliver hydrophilic and hydrophobiccompounds such as, but not limited to, polynucleotides, primaryconstructs and/or mmRNA. In one aspect, the oncology-relatedpolynucleotides, primary constructs and/or mmRNA disclosed herein areformulated in a NanoLiposome such as, but not limited to, CeramideNanoLiposomes.

Excipients

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference in its entirety) disclosesvarious excipients used in formulating pharmaceutical compositions andknown techniques for the preparation thereof. Except insofar as anyconventional excipient medium is incompatible with a substance or itsderivatives, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical compositions.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEENn®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate[SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfate, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Delivery

The present disclosure encompasses the delivery of oncology-relatedpolynucleotides, primary constructs or mmRNA for any of therapeutic,pharmaceutical, diagnostic or imaging by any appropriate route takinginto consideration likely advances in the sciences of drug delivery.Delivery may be naked or formulated.

Naked Delivery

The oncology-related polynucleotides, primary constructs or mmRNA of thepresent invention may be delivered to a cell naked. As used herein in,“naked” refers to delivering polynucleotides, primary constructs ormmRNA free from agents which promote transfection. For example, theoncology-related polynucleotides, primary constructs or mmRNA deliveredto the cell may contain no modifications. The naked polynucleotides,primary constructs or mmRNA may be delivered to the cell using routes ofadministration known in the art and described herein.

Formulated Delivery

The oncology-related polynucleotides, primary constructs or mmRNA of thepresent invention may be formulated, using the methods described herein.The formulations may contain polynucleotides, primary constructs ormmRNA which may be modified and/or unmodified. The formulations mayfurther include, but are not limited to, cell penetration agents, apharmaceutically acceptable carrier, a delivery agent, a bioerodible orbiocompatible polymer, a solvent, and a sustained-release deliverydepot. The formulated polynucleotides, primary constructs or mmRNA maybe delivered to the cell using routes of administration known in the artand described herein.

The compositions may also be formulated for direct delivery to an organor tissue in any of several ways in the art including, but not limitedto, direct soaking or bathing, via a catheter, by gels, powder,ointments, creams, gels, lotions, and/or drops, by using substrates suchas fabric or biodegradable materials coated or impregnated with thecompositions, and the like.

Administration

The oncology-related polynucleotides, primary constructs or mmRNA of thepresent invention may be administered by any route which results in atherapeutically effective outcome. These include, but are not limited toenteral, gastroenteral, epidural, oral, transdermal, epidural(peridural), intracerebral (into the cerebrum), intracerebroventricular(into the cerebral ventricles), epicutaneous (application onto theskin), intradermal, (into the skin itself), subcutaneous (under theskin), nasal administration (through the nose), intravenous (into avein), intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection, (into the base of thepenis), intravaginal administration, intrauterine, extra-amnioticadministration, transdermal (diffusion through the intact skin forsystemic distribution), transmucosal (diffusion through a mucousmembrane), insufflation (snorting), sublingual, sublabial, enema, eyedrops (onto the conjunctiva), or in ear drops. In specific embodiments,compositions may be administered in a way which allows them cross theblood-brain barrier, vascular barrier, or other epithelial barrier.Non-limiting routes of administration for the oncology-relatedpolynucleotides, primary constructs or mmRNA of the present inventionare described below.

Parenteral and Injectible Administration

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, oral compositions can include adjuvants such as wettingagents, emulsifying and suspending agents, sweetening, flavoring, and/orperfuming agents. In certain embodiments for parenteral administration,compositions are mixed with solubilizing agents such as CREMOPHOR®,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Oral Administration

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups, and/or elixirs. In addition to active ingredients,liquid dosage forms may comprise inert diluents commonly used in the artsuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and/or perfuming agents.In certain embodiments for parenteral administration, compositions aremixed with solubilizing agents such as CREMOPHOR®, alcohols, oils,modified oils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g. starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g. agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g. paraffin), absorptionaccelerators (e.g. quaternary ammonium compounds), wetting agents (e.g.cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin andbentonite clay), and lubricants (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate), andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may comprise buffering agents.

Topical or Transdermal Administration

As described herein, compositions containing the oncology-relatedpolynucleotides, primary constructs or mmRNA of the invention may beformulated for administration topically. The skin may be an ideal targetsite for delivery as it is readily accessible. Gene expression may berestricted not only to the skin, potentially avoiding nonspecifictoxicity, but also to specific layers and cell types within the skin.

The site of cutaneous expression of the delivered compositions willdepend on the route of nucleic acid delivery. Three routes are commonlyconsidered to deliver polynucleotides, primary constructs or mmRNA tothe skin: (i) topical application (e.g. for local/regional treatmentand/or oncology-related applications); (ii) intradermal injection (e.g.for local/regional treatment and/or oncology-related applications); and(iii) systemic delivery (e.g. for treatment of dermatologic diseasesthat affect both cutaneous and extracutaneous regions). Polynucleotides,primary constructs or mmRNA can be delivered to the skin by severaldifferent approaches known in the art. Most topical delivery approacheshave been shown to work for delivery of DNA, such as but not limited to,topical application of non-cationic liposome-DNA complex, cationicliposome-DNA complex, particle-mediated (gene gun), puncture-mediatedgene transfections, and viral delivery approaches. After delivery of thenucleic acid, gene products have been detected in a number of differentskin cell types, including, but not limited to, basal keratinocytes,sebaceous gland cells, dermal fibroblasts and dermal macrophages.

In one embodiment, the invention provides for a variety of dressings(e.g., wound dressings) or bandages (e.g., adhesive bandages) forconveniently and/or effectively carrying out methods of the presentinvention. Typically dressing or bandages may comprise sufficientamounts of pharmaceutical compositions and/or polynucleotides, primaryconstructs or mmRNA described herein to allow a user to perform multipletreatments of a subject(s).

In one embodiment, the invention provides for the oncology-relatedpolynucleotides, primary constructs or mmRNA compositions to bedelivered in more than one injection.

In one embodiment, before topical and/or transdermal administration atleast one area of tissue, such as skin, may be subjected to a deviceand/or solution which may increase permeability. In one embodiment, thetissue may be subjected to an abrasion device to increase thepermeability of the skin (see U.S. Patent Publication No. 20080275468,herein incorporated by reference in its entirety). In anotherembodiment, the tissue may be subjected to an ultrasound enhancementdevice. An ultrasound enhancement device may include, but is not limitedto, the devices described in U.S. Publication No. 20040236268 and U.S.Pat. Nos. 6,491,657 and 6,234,990; each of which are herein incorporatedby reference in their entireties. Methods of enhancing the permeabilityof tissue are described in U.S. Publication Nos. 20040171980 and20040236268 and U.S. Pat. No. 6,190,315; each of which are hereinincorporated by reference in their entireties.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein.The permeability of skin may be measured by methods known in the artand/or described in U.S. Pat. No. 6,190,315, herein incorporated byreference in its entirety. As a non-limiting example, a modified mRNAformulation may be delivered by the drug delivery methods described inU.S. Pat. No. 6,190,315, herein incorporated by reference in itsentirety.

In another non-limiting example tissue may be treated with a eutecticmixture of local anesthetics (EMLA) cream before, during and/or afterthe tissue may be subjected to a device which may increase permeability.Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated byreference in its entirety) showed that using the EMLA cream incombination with a low energy, an onset of superficial cutaneousanalgesia was seen as fast as 5 minutes after a pretreatment with a lowenergy ultrasound.

In one embodiment, enhancers may be applied to the tissue before,during, and/or after the tissue has been treated to increasepermeability. Enhancers include, but are not limited to, transportenhancers, physical enhancers, and cavitation enhancers. Non-limitingexamples of enhancers are described in U.S. Pat. No. 6,190,315, hereinincorporated by reference in its entirety.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein,which may further contain a substance that invokes an immune response.In another non-limiting example, a formulation containing a substance toinvoke an immune response may be delivered by the methods described inU.S. Publication Nos. 20040171980 and 20040236268; each of which areherein incorporated by reference in their entireties.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present inventioncontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.

Topically-administrable formulations may, for example, comprise fromabout 0.1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Depot Administration

As described herein, in some embodiments, the composition is formulatedin depots for extended release. Generally, a specific organ or tissue (a“target tissue”) is targeted for administration.

In some aspects of the invention, the oncology-related polynucleotides,primary constructs or mmRNA are spatially retained within or proximal toa target tissue. Provided are method of providing a composition to atarget tissue of a mammalian subject by contacting the target tissue(which contains one or more target cells) with the composition underconditions such that the composition, in particular the nucleic acidcomponent(s) of the composition, is substantially retained in the targettissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90,95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of thecomposition is retained in the target tissue. Advantageously, retentionis determined by measuring the amount of the nucleic acid present in thecomposition that enters one or more target cells. For example, at least1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9,99.99 or greater than 99.99% of the nucleic acids administered to thesubject are present intracellularly at a period of time followingadministration. For example, intramuscular injection to a mammaliansubject is performed using an aqueous composition containing aribonucleic acid and a transfection reagent, and retention of thecomposition is determined by measuring the amount of the ribonucleicacid present in the muscle cells.

Aspects of the invention are directed to methods of providing acomposition to a target tissue of a mammalian subject, by contacting thetarget tissue (containing one or more target cells) with the compositionunder conditions such that the composition is substantially retained inthe target tissue. The composition contains an effective amount of apolynucleotides, primary constructs or mmRNA such that the polypeptideof interest is produced in at least one target cell. The compositionsgenerally contain a cell penetration agent, although “naked” nucleicacid (such as nucleic acids without a cell penetration agent or otheragent) is also contemplated, and a pharmaceutically acceptable carrier.

In some circumstances, the amount of a protein produced by cells in atissue is desirably increased. Preferably, this increase in proteinproduction is spatially restricted to cells within the target tissue.Thus, provided are methods of increasing production of a protein ofinterest in a tissue of a mammalian subject. A composition is providedthat contains polynucleotides, primary constructs or mmRNA characterizedin that a unit quantity of composition has been determined to producethe polypeptide of interest in a substantial percentage of cellscontained within a predetermined volume of the target tissue.

In some embodiments, the composition includes a plurality of differentpolynucleotides, primary constructs or mmRNA, where one or more than oneof the oncology-related polynucleotides, primary constructs or mmRNAencodes a polypeptide of interest. Optionally, the composition alsocontains a cell penetration agent to assist in the intracellulardelivery of the composition. A determination is made of the dose of thecomposition required to produce the polypeptide of interest in asubstantial percentage of cells contained within the predeterminedvolume of the target tissue (generally, without inducing significantproduction of the polypeptide of interest in tissue adjacent to thepredetermined volume, or distally to the target tissue). Subsequent tothis determination, the determined dose is introduced directly into thetissue of the mammalian subject.

In one embodiment, the invention provides for the oncology-relatedpolynucleotides, primary constructs or mmRNA to be delivered in morethan one injection or by split dose injections.

In one embodiment, the invention may be retained near target tissueusing a small disposable drug reservoir, patch pump or osmotic pump.Non-limiting examples of patch pumps include those manufactured and/orsold by BD® (Franklin Lakes, N.J.), Insulet Corporation (Bedford,Mass.), SteadyMed Therapeutics (San Francisco, Calif.), Medtronic(Minneapolis, Minn.) (e.g., MiniMed), UniLife (York, Pa.), Valeritas(Bridgewater, N.J.), and SpringLeaf Therapeutics (Boston, Mass.). Anon-limiting example of an osmotic pump include those manufactured byDURECT® (Cupertino, Calif.) (e.g., DUROS® and ALZET®).

Pulmonary Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions aresuitably in the form of dry powders for administration using a devicecomprising a dry powder reservoir to which a stream of propellant may bedirected to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further comprise additional ingredients such as a liquidnon-ionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles comprising theactive ingredient).

As a non-limiting example, the oncology-related polynucleotides, primaryconstructs and/or mmRNA described herein may be formulated for pulmonarydelivery by the methods described in U.S. Pat. No. 8,257,685; hereinincorporated by reference in its entirety.

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Intranasal, Nasal and Buccal Administration

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

Ophthalmic Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis invention. A multilayer thin film device may be prepared to containa pharmaceutical composition for delivery to the eye and/or surroundingtissue.

Payload Administration: Detectable Agents and Therapeutic Agents

The oncology-related polynucleotides, primary constructs or mmRNAdescribed herein can be used in a number of different scenarios in whichdelivery of a substance (the “payload”) to a biological target isdesired, for example delivery of detectable substances for detection ofthe target, or delivery of a therapeutic agent. Detection methods caninclude, but are not limited to, both imaging in vitro and in vivoimaging methods, e.g., immunohistochemistry, bioluminescence imaging(BLI), Magnetic Resonance Imaging (MRI), positron emission tomography(PET), electron microscopy, X-ray computed tomography, Raman imaging,optical coherence tomography, absorption imaging, thermal imaging,fluorescence reflectance imaging, fluorescence microscopy, fluorescencemolecular tomographic imaging, nuclear magnetic resonance imaging, X-rayimaging, ultrasound imaging, photoacoustic imaging, lab assays, or inany situation where tagging/staining/imaging is required.

The oncology-related polynucleotides, primary constructs or mmRNA can bedesigned to include both a linker and a payload in any usefulorientation. For example, a linker having two ends is used to attach oneend to the payload and the other end to the nucleobase, such as at theC-7 or C-8 positions of the deaza-adenosine or deaza-guanosine or to theN-3 or C-5 positions of cytosine or uracil. The polynucleotide of theinvention can include more than one payload (e.g., a label and atranscription inhibitor), as well as a cleavable linker. In oneembodiment, the modified nucleotide is a modified 7-deaza-adenosinetriphosphate, where one end of a cleavable linker is attached to the C7position of 7-deaza-adenine, the other end of the linker is attached toan inhibitor (e.g., to the C5 position of the nucleobase on a cytidine),and a label (e.g., Cy5) is attached to the center of the linker (see,e.g., compound 1 of A*pCp C5 Parg Capless in FIG. 5 and columns 9 and 10of U.S. Pat. No. 7,994,304, incorporated herein by reference). Uponincorporation of the modified 7-deaza-adenosine triphosphate to anencoding region, the resulting polynucleotide having a cleavable linkerattached to a label and an inhibitor (e.g., a polymerase inhibitor).Upon cleavage of the linker (e.g., with reductive conditions to reduce alinker having a cleavable disulfide moiety), the label and inhibitor arereleased. Additional linkers and payloads (e.g., therapeutic agents,detectable labels, and cell penetrating payloads) are described herein.

Scheme 12 below depicts an exemplary modified nucleotide wherein thenucleobase, adenine, is attached to a linker at the C-7 carbon of7-deaza adenine. In addition, Scheme 12 depicts the modified nucleotidewith the linker and payload, e.g., a detectable agent, incorporated ontothe 3′ end of the mRNA. Disulfide cleavage and 1,2-addition of the thiolgroup onto the propargyl ester releases the detectable agent. Theremaining structure (depicted, for example, as pApC5Parg in Scheme 12)is the inhibitor. The rationale for the structure of the modifiednucleotides is that the tethered inhibitor sterically interferes withthe ability of the polymerase to incorporate a second base. Thus, it iscritical that the tether be long enough to affect this function and thatthe inhibitor be in a stereochemical orientation that inhibits orprohibits second and follow on nucleotides into the growingpolynucleotide strand.

For example, the oncology-related polynucleotides, primary constructs ormmRNA described herein can be used in reprogramming induced pluripotentstem cells (iPS cells), which can directly track cells that aretransfected compared to total cells in the cluster. In another example,a drug that may be attached to the oncology-related polynucleotides,primary constructs or mmRNA via a linker and may be fluorescentlylabeled can be used to track the drug in vivo, e.g. intracellularly.Other examples include, but are not limited to, the use of apolynucleotides, primary constructs or mmRNA in reversible drug deliveryinto cells.

The oncology-related polynucleotides, primary constructs or mmRNAdescribed herein can be used in intracellular targeting of a payload,e.g., detectable or therapeutic agent, to specific organelle. Exemplaryintracellular targets can include, but are not limited to, the nuclearlocalization for advanced mRNA processing, or a nuclear localizationsequence (NLS) linked to the mRNA containing an inhibitor.

In addition, the oncology-related polynucleotides, primary constructs ormmRNA described herein can be used to deliver therapeutic agents tocells or tissues, e.g., in living animals. For example, theoncology-related polynucleotides, primary constructs or mmRNA describedherein can be used to deliver highly polar chemotherapeutics agents tokill cancer cells. The oncology-related polynucleotides, primaryconstructs or mmRNA attached to the therapeutic agent through a linkercan facilitate member permeation allowing the therapeutic agent totravel into a cell to reach an intracellular target.

In one example, the linker is attached at the 2′-position of the ribosering and/or at the 3′ and/or 5′ position of the polynucleotides, primaryconstructs mmRNA (See e.g., International Pub. No. WO2012030683, hereinincorporated by reference in its entirety). The linker may be any linkerdisclosed herein, known in the art and/or disclosed in InternationalPub. No. WO2012030683, herein incorporated by reference in its entirety.

In another example, the oncology-related polynucleotides, primaryconstructs or mmRNA can be attached to the oncology-relatedpolynucleotides, primary constructs or mmRNA a viral inhibitory peptide(VIP) through a cleavable linker. The cleavable linker can release theVIP and dye into the cell. In another example, the oncology-relatedpolynucleotides, primary constructs or mmRNA can be attached through thelinker to an ADP-ribosylate, which is responsible for the actions ofsome bacterial toxins, such as cholera toxin, diphtheria toxin, andpertussis toxin. These toxin proteins are ADP-ribosyltransferases thatmodify target proteins in human cells. For example, cholera toxinADP-ribosylates G proteins modifies human cells by causing massive fluidsecretion from the lining of the small intestine, which results inlife-threatening diarrhea.

In some embodiments, the payload may be a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that may bedetrimental to cells. Examples include, but are not limited to, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, teniposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein inits entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,5,585,499, and 5,846,545, all of which are incorporated herein byreference), and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

In some embodiments, the payload may be a detectable agent, such asvarious organic small molecules, inorganic compounds, nanoparticles,enzymes or enzyme substrates, fluorescent materials, luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin, and aequorin), chemiluminescent materials, radioactivematerials (e.g., ¹⁸F, ⁶⁷Ga, ^(81m)Kr, ⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl,¹²⁵I, ³⁵S, ¹⁴C, ³H, or ^(99m)Tc (e.g., as pertechnetate(technetate(VII), TcO₄ ⁻)), and contrast agents (e.g., gold (e.g., goldnanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g.,superparamagnetic iron oxide (SPIO), monocrystalline iron oxidenanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide(USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinatedcontrast media (iohexol), microbubbles, or perfluorocarbons). Suchoptically-detectable labels include for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and -6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodarnine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolicacid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable agent may be a non-detectableprecursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Invitro assays in which the enzyme labeled compositions can be usedinclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), immunoprecipitation assays, immunofluorescence, enzymeimmunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.

Combinations

The oncology-related polynucleotides, primary constructs or mmRNA may beused in combination with one or more other therapeutic, prophylactic,diagnostic, or imaging agents. By “in combination with,” it is notintended to imply that the agents must be administered at the same timeand/or formulated for delivery together, although these methods ofdelivery are within the scope of the present disclosure. Compositionscan be administered concurrently with, prior to, or subsequent to, oneor more other desired therapeutics or medical procedures. In general,each agent will be administered at a dose and/or on a time scheduledetermined for that agent. In some embodiments, the present disclosureencompasses the delivery of pharmaceutical, prophylactic, diagnostic, orimaging compositions in combination with agents that may improve theirbioavailability, reduce and/or modify their metabolism, inhibit theirexcretion, and/or modify their distribution within the body. As anon-limiting example, the nucleic acids or mmRNA may be used incombination with a pharmaceutical agent for the treatment of cancer orto control hyperproliferative cells. In U.S. Pat. No. 7,964,571, hereinincorporated by reference in its entirety, a combination therapy for thetreatment of solid primary or metastasized tumor is described using apharmaceutical composition including a DNA plasmid encoding forinterleukin-12 with a lipopolymer and also administering at least oneanticancer agent or chemotherapeutic. Further, the nucleic acids andmmRNA of the present invention that encodes anti-proliferative moleculesmay be in a pharmaceutical composition with a lipopolymer (see e.g.,U.S. Pub. No. 20110218231, herein incorporated by reference in itsentirety, claiming a pharmaceutical composition comprising a DNA plasmidencoding an anti-proliferative molecule and a lipopolymer) which may beadministered with at least one chemotherapeutic or anticancer agent.

It will further be appreciated that therapeutically, prophylactically,diagnostically, or imaging active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, it is expected that agentsutilized in combination with be utilized at levels that do not exceedthe levels at which they are utilized individually. In some embodiments,the levels utilized in combination will be lower than those utilizedindividually. In one embodiment, the combinations, each or together maybe administered according to the split dosing regimens described herein.

Dosing

The present invention provides methods comprising administering modifiedmRNAs and their encoded proteins or complexes in accordance with theinvention to a subject in need thereof. Nucleic acids, proteins orcomplexes, or pharmaceutical, imaging, diagnostic, or prophylacticcompositions thereof, may be administered to a subject using any amountand any route of administration effective for preventing, treating,diagnosing, or imaging a disease, disorder, and/or condition (e.g., adisease, disorder, and/or condition relating to working memorydeficits). The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Compositions inaccordance with the invention are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention may be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect. The desired dosage may be deliveredthree times a day, two times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used.

According to the present invention, it has been discovered thatadministration of mmRNA in split-dose regimens produce higher levels ofproteins in mammalian subjects. As used herein, a “split dose” is thedivision of single unit dose or total daily dose into two or more doses,e.g, two or more administrations of the single unit dose. As usedherein, a “single unit dose” is a dose of any therapeutic administeredin one dose/at one time/single route/single point of contact, i.e.,single administration event. As used herein, a “total daily dose” is anamount given or prescribed in 24 hr period. It may be administered as asingle unit dose. In one embodiment, the mmRNA of the present inventionare administered to a subject in split doses. The mmRNA may beformulated in buffer only or in a formulation described herein.

Dosage Forms

A pharmaceutical composition described herein can be formulated into adosage form described herein, such as a topical, intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intracardiac, intraperitoneal,subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art including, but not limited to, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. In certainembodiments for parenteral administration, compositions may be mixedwith solubilizing agents such as CREMOPHOR®, alcohols, oils, modifiedoils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known art andmay include suitable dispersing agents, wetting agents, and/orsuspending agents. Sterile injectable preparations may be sterileinjectable solutions, suspensions, and/or emulsions in nontoxicparenterally acceptable diluents and/or solvents, for example, asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed include, but are not limited to, water, Ringer'ssolution, U.S.P., and isotonic sodium chloride solution. Sterile, fixedoils are conventionally employed as a solvent or suspending medium. Forthis purpose any bland fixed oil can be employed including syntheticmono- or diglycerides. Fatty acids such as oleic acid can be used in thepreparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it may bedesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the polynucleotide,primary construct or mmRNA then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administeredpolynucleotide, primary construct or mmRNA may be accomplished bydissolving or suspending the polynucleotide, primary construct or mmRNAin an oil vehicle. Injectable depot forms are made by formingmicroencapsule matrices of the polynucleotide, primary construct ormmRNA in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of polynucleotide, primary construct or mmRNAto polymer and the nature of the particular polymer employed, the rateof polynucleotide, primary construct or mmRNA release can be controlled.Examples of other biodegradable polymers include, but are not limitedto, poly(orthoesters) and poly(anhydrides). Depot injectableformulations may be prepared by entrapping the polynucleotide, primaryconstruct or mmRNA in liposomes or microemulsions which are compatiblewith body tissues.

Pulmonary

Formulations described herein as being useful for pulmonary delivery mayalso be used for intranasal delivery of a pharmaceutical composition.Another formulation suitable for intranasal administration may be acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 μm to 500 μm. Such a formulation may beadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, contain about 0.1% to 20% (w/w) active ingredient, where thebalance may comprise an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

Properties of Pharmaceutical Compositions

The pharmaceutical compositions described herein can be characterized byone or more of bioavailability, therapeutic window and/or volume ofdistribution.

Bioavailability

The oncology-related polynucleotides, primary constructs or mmRNA, whenformulated into a composition with a delivery agent as described herein,can exhibit an increase in bioavailability as compared to a compositionlacking a delivery agent as described herein. As used herein, the term“bioavailability” refers to the systemic availability of a given amountof oncology-related polynucleotides, primary constructs or mmRNAadministered to a mammal. Bioavailability can be assessed by measuringthe area under the curve (AUC) or the maximum serum or plasmaconcentration (C_(max)) of the unchanged form of a compound followingadministration of the compound to a mammal. AUC is a determination ofthe area under the curve plotting the serum or plasma concentration of acompound along the ordinate (Y-axis) against time along the abscissa(X-axis). Generally, the AUC for a particular compound can be calculatedusing methods known to those of ordinary skill in the art and asdescribed in G. S. Banker, Modern Pharmaceutics, Drugs and thePharmaceutical Sciences, v. 72, Marcel Dekker, New York, Inc., 1996,herein incorporated by reference in its entirety.

The C_(max) value is the maximum concentration of the compound achievedin the serum or plasma of a mammal following administration of thecompound to the mammal. The C_(max) value of a particular compound canbe measured using methods known to those of ordinary skill in the art.The phrases “increasing bioavailability” or “improving thepharmacokinetics,” as used herein mean that the systemic availability ofa first polynucleotide, primary construct or mmRNA, measured as AUC,C_(max), or C_(min) in a mammal is greater, when co-administered with adelivery agent as described herein, than when such co-administrationdoes not take place. In some embodiments, the bioavailability of thepolynucleotide, primary construct or mmRNA can increase by at leastabout 2%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%.

Therapeutic Window

The oncology-related polynucleotides, primary constructs or mmRNA, whenformulated into a composition with a delivery agent as described herein,can exhibit an increase in the therapeutic window of the administeredpolynucleotide, primary construct or mmRNA composition as compared tothe therapeutic window of the administered polynucleotide, primaryconstruct or mmRNA composition lacking a delivery agent as describedherein. As used herein “therapeutic window” refers to the range ofplasma concentrations, or the range of levels of therapeutically activesubstance at the site of action, with a high probability of eliciting atherapeutic effect. In some embodiments, the therapeutic window of thepolynucleotide, primary construct or mmRNA when co-administered with adelivery agent as described herein can increase by at least about 2%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or about 100%.

Volume of Distribution

The oncology-related polynucleotides, primary constructs or mmRNA, whenformulated into a composition with a delivery agent as described herein,can exhibit an improved volume of distribution (V_(dist)), e.g., reducedor targeted, relative to a composition lacking a delivery agent asdescribed herein. The volume of distribution (V_(dist)) relates theamount of the drug in the body to the concentration of the drug in theblood or plasma. As used herein, the term “volume of distribution”refers to the fluid volume that would be required to contain the totalamount of the drug in the body at the same concentration as in the bloodor plasma: V_(dist) equals the amount of drug in the body/concentrationof drug in blood or plasma. For example, for a 10 mg dose and a plasmaconcentration of 10 mg/L, the volume of distribution would be 1 liter.The volume of distribution reflects the extent to which the drug ispresent in the extravascular tissue. A large volume of distributionreflects the tendency of a compound to bind to the tissue componentscompared with plasma protein binding. In a clinical setting, V_(dist)can be used to determine a loading dose to achieve a steady stateconcentration. In some embodiments, the volume of distribution of thepolynucleotide, primary construct or mmRNA when co-administered with adelivery agent as described herein can decrease at least about 2%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%.

Biological Effect

In one embodiment, the biological effect of the modified mRNA deliveredto the animals may be categorized by analyzing the protein expression inthe animals. The protein expression may be determined from analyzing abiological sample collected from a mammal administered the modified mRNAof the present invention. In one embodiment, the expression proteinencoded by the modified mRNA administered to the mammal of at least 50pg/ml may be preferred. For example, a protein expression of 50-200pg/ml for the protein encoded by the modified mRNA delivered to themammal may be seen as a therapeutically effective amount of protein inthe mammal.

Detection of Modified Nucleic Acids by Mass Spectrometry

Mass spectrometry (MS) is an analytical technique that can providestructural and molecular mass/concentration information on moleculesafter their conversion to ions. The molecules are first ionized toacquire positive or negative charges and then they travel through themass analyzer to arrive at different areas of the detector according totheir mass/charge (m/z) ratio.

Mass spectrometry is performed using a mass spectrometer which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. For example ionization of the sample maybe performed by electrospray ionization (ESI), atmospheric pressurechemical ionization (APCI), photoionization, electron ionization, fastatom bombardment (FAB)/liquid secondary ionization (LSIMS), matrixassisted laser desorption/ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, and particle beamionization. The skilled artisan will understand that the choice ofionization method can be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Suitable analyzers for determiningmass-to-charge ratios include quadropole analyzers, ion traps analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM).

Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupledwith stable isotope labeled dilution of peptide standards has been shownto be an effective method for protein verification (e.g., Keshishian etal., Mol Cell Proteomics 2009 8: 2339-2349; Kuhn et al., Clin Chem 200955:1108-1117; Lopez et al., Clin Chem 2010 56:281-290; each of which areherein incorporated by reference in its entirety). Unlike untargetedmass spectrometry frequently used in biomarker discovery studies,targeted MS methods are peptide sequence-based modes of MS that focusthe full analytical capacity of the instrument on tens to hundreds ofselected peptides in a complex mixture. By restricting detection andfragmentation to only those peptides derived from proteins of interest,sensitivity and reproducibility are improved dramatically compared todiscovery-mode MS methods. This method of mass spectrometry-basedmultiple reaction monitoring (MRM) quantitation of proteins candramatically impact the discovery and quantitation of biomarkers viarapid, targeted, multiplexed protein expression profiling of clinicalsamples.

In one embodiment, a biological sample which may contain at least oneprotein encoded by at least one modified mRNA of the present inventionmay be analyzed by the method of MRM-MS. The quantification of thebiological sample may further include, but is not limited to,isotopically labeled peptides or proteins as internal standards.

According to the present invention, the biological sample, once obtainedfrom the subject, may be subjected to enzyme digestion. As used herein,the term “digest” means to break apart into shorter peptides. As usedherein, the phrase “treating a sample to digest proteins” meansmanipulating a sample in such a way as to break down proteins in asample. These enzymes include, but are not limited to, trypsin,endoproteinase Glu-C and chymotrypsin. In one embodiment, a biologicalsample which may contain at least one protein encoded by at least onemodified mRNA of the present invention may be digested using enzymes.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for proteinusing electrospray ionization. Electrospray ionization (ESI) massspectrometry (ESIMS) uses electrical energy to aid in the transfer ofions from the solution to the gaseous phase before they are analyzed bymass spectrometry. Samples may be analyzed using methods known in theart (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12; hereinincorporated by reference in its entirety). The ionic species containedin solution may be transferred into the gas phase by dispersing a finespray of charge droplets, evaporating the solvent and ejecting the ionsfrom the charged droplets to generate a mist of highly charged droplets.The mist of highly charged droplets may be analyzed using at least 1, atleast 2, at least 3 or at least 4 mass analyzers such as, but notlimited to, a quadropole mass analyzer. Further, the mass spectrometrymethod may include a purification step. As a non-limiting example, thefirst quadrapole may be set to select a single m/z ratio so it mayfilter out other molecular ions having a different m/z ratio which mayeliminate complicated and time-consuming sample purification proceduresprior to MS analysis.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for protein ina tandem ESIMS system (e.g., MS/MS). As non-limiting examples, thedroplets may be analyzed using a product scan (or daughter scan) aprecursor scan (parent scan) a neutral loss or a multiple reactionmonitoring.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed usingmatrix-assisted laser desorption/ionization (MALDI) mass spectrometry(MALDIMS). MALDI provides for the nondestructive vaporization andionization of both large and small molecules, such as proteins. In MALDIanalysis, the analyte is first co-crystallized with a large molar excessof a matrix compound, which may also include, but is not limited to, anultraviolet absorbing weak organic acid. Non-limiting examples ofmatrices used in MALDI are α-cyano-4-hydroxycinnamic acid,3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.Laser radiation of the analyte-matrix mixture may result in thevaporization of the matrix and the analyte. The laser induced desorptionprovides high ion yields of the intact analyte and allows formeasurement of compounds with high accuracy. Samples may be analyzedusing methods known in the art (e.g., Lewis, Wei and Siuzdak,Encyclopedia of Analytical Chemistry 2000:5880-5894; herein incorporatedby reference in its entirety). As non-limiting examples, mass analyzersused in the MALDI analysis may include a linear time-of-flight (TOF), aTOF reflectron or a Fourier transform mass analyzer.

In one embodiment, the analyte-matrix mixture may be formed using thedried-droplet method. A biologic sample is mixed with a matrix to createa saturated matrix solution where the matrix-to-sample ratio isapproximately 5000:1. An aliquot (approximately 0.5-2.0 uL) of thesaturated matrix solution is then allowed to dry to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethin-layer method. A matrix homogeneous film is first formed and thenthe sample is then applied and may be absorbed by the matrix to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethick-layer method. A matrix homogeneous film is formed with anitro-cellulose matrix additive. Once the uniform nitro-cellulose matrixlayer is obtained the sample is applied and absorbed into the matrix toform the analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thesandwich method. A thin layer of matrix crystals is prepared as in thethin-layer method followed by the addition of droplets of aqueoustrifluoroacetic acid, the sample and matrix. The sample is then absorbedinto the matrix to form the analyte-matrix mixture.

V. USES OF ONCOLOGY-RELATED POLYNUCLEOTIDES, ONCOLOGY-RELATED PRIMARYCONSTRUCTS AND ONCOLOGY-RELATED MMRNA OF THE INVENTION

The oncology-related polynucleotides, oncology-related primaryconstructs and oncology-related mmRNA of the present invention aredesigned, in preferred embodiments, to provide for avoidance or evasionof deleterious bio-responses such as the immune response and/ordegradation pathways, overcoming the threshold of expression and/orimproving protein production capacity, improved expression rates ortranslation efficiency, improved drug or protein half life and/orprotein concentrations, optimized protein localization, to improve oneor more of the stability and/or clearance in tissues, receptor uptakeand/or kinetics, cellular access by the compositions, engagement withtranslational machinery, secretion efficiency (when applicable),accessibility to circulation, and/or modulation of a cell's status,function and/or activity.

Therapeutics Therapeutic Agents

The oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA of the present invention, such asmodified nucleic acids and modified RNAs, and the proteins translatedfrom them described herein can be used as therapeutic or prophylacticagents. They are provided for use in medicine. For example, anoncology-related polynucleotide, oncology-related primary construct oroncology-related mmRNA described herein can be administered to asubject, wherein the oncology-related polynucleotide, oncology-relatedprimary construct or oncology-related mmRNA is translated in vivo toproduce a therapeutic or prophylactic oncology-related polypeptide inthe subject. Provided are compositions, methods, kits, and reagents fordiagnosis, treatment or prevention of a disease or condition in humansand other mammals. The active therapeutic agents of the inventioninclude oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA, cells containing oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA or polypeptides translated from the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA.

In certain embodiments, provided herein are combination therapeuticscontaining one or more oncology-related polynucleotide, oncology-relatedprimary construct or oncology-related mmRNA containing translatableregions that encode for a protein or proteins that boost a mammaliansubject's immunity along with a protein that induces antibody-dependentcellular toxicity. For example, provided herein are therapeuticscontaining one or more nucleic acids that encode trastuzumab andgranulocyte-colony stimulating factor (G-CSF). In particular, suchcombination therapeutics are useful in Her2+ breast cancer patients whodevelop induced resistance to trastuzumab. (See, e.g., Albrecht,Immunotherapy. 2(6):795-8 (2010)).

Provided herein are methods of inducing translation of a recombinantpolypeptide in a cell population using the oncology-relatedpolynucleotide, oncology-related primary construct or oncology-relatedmmRNA described herein. Such translation can be in vivo, ex vivo, inculture, or in vitro. The cell population is contacted with an effectiveamount of a composition containing an oncology-related nucleic acid thathas at least one nucleoside modification, and a translatable regionencoding the recombinant oncology-related polypeptide. The population iscontacted under conditions such that the oncology-related nucleic acidis localized into one or more cells of the cell population and therecombinant oncology-related polypeptide is translated in the cell fromthe oncology-related nucleic acid.

An “effective amount” of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the nucleic acid (e.g., size, and extent ofmodified nucleosides), and other determinants. In general, an effectiveamount of the composition provides efficient protein production in thecell, preferably more efficient than a composition containing acorresponding unmodified nucleic acid. Increased efficiency may bedemonstrated by increased cell transfection (i.e., the percentage ofcells transfected with the nucleic acid), increased protein translationfrom the nucleic acid, decreased nucleic acid degradation (asdemonstrated, e.g., by increased duration of protein translation from amodified nucleic acid), or reduced innate immune response of the hostcell.

Aspects of the invention are directed to methods of inducing in vivotranslation of a recombinant polypeptide in a mammalian subject in needthereof. Therein, an effective amount of a composition containing anucleic acid that has at least one structural or chemical modificationand a translatable region encoding the recombinant polypeptide isadministered to the subject using the delivery methods described herein.The nucleic acid is provided in an amount and under other conditionssuch that the nucleic acid is localized into a cell of the subject andthe recombinant polypeptide is translated in the cell from the nucleicacid. The cell in which the nucleic acid is localized, or the tissue inwhich the cell is present, may be targeted with one or more than onerounds of nucleic acid administration.

In certain embodiments, the administered oncology-relatedpolynucleotide, oncology-related primary construct or oncology-relatedmmRNA directs production of one or more recombinant polypeptides thatprovide a functional activity which is substantially absent in the cell,tissue or organism in which the recombinant oncology-related polypeptideis translated. For example, the missing functional activity may beenzymatic, structural, or gene regulatory in nature. In relatedembodiments, the administered oncology-related polynucleotide,oncology-related primary construct or oncology-related mmRNA directsproduction of one or more recombinant oncology-related polypeptides thatincreases (e.g., synergistically) a functional activity which is presentbut substantially deficient in the cell in which the recombinantoncology-related polypeptide is translated.

In other embodiments, the administered oncology-related polynucleotide,oncology-related primary construct or oncology-related mmRNA directsproduction of one or more recombinant oncology-related polypeptides thatreplace an oncology-related polypeptide (or multiple oncology-relatedpolypeptides) that is substantially absent in the cell in which therecombinant oncology-related polypeptide is translated. Such absence maybe due to genetic mutation of the encoding gene or regulatory pathwaythereof. In some embodiments, the recombinant oncology-relatedpolypeptide increases the level of an endogenous oncology-relatedprotein in the cell to a desirable level; such an increase may bring thelevel of the endogenous oncology-related protein from a subnormal levelto a normal level or from a normal level to a super-normal level.

Alternatively, the recombinant oncology-related polypeptide functions toantagonize the activity of an endogenous protein present in, on thesurface of, or secreted from the cell. Usually, the activity of theendogenous oncology-related protein is deleterious to the subject; forexample, due to mutation of the endogenous oncology-related proteinresulting in altered activity or localization. Additionally, therecombinant oncology-related polypeptide antagonizes, directly orindirectly, the activity of a biological moiety present in, on thesurface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a nucleic acid, a carbohydrate, aprotein toxin such as shiga and tetanus toxins, or a small moleculetoxin such as botulinum, cholera, and diphtheria toxins. Additionally,the antagonized biological molecule may be an endogenous protein thatexhibits an undesirable activity, such as a cytotoxic or cytostaticactivity.

The recombinant oncology-related proteins described herein may beengineered for localization within the cell, potentially within aspecific compartment such as the nucleus, or are engineered forsecretion from the cell or translocation to the plasma membrane of thecell.

In some embodiments, modified oncology-related mRNAs and their encodedoncology-related polypeptides in accordance with the present inventionmay be used for treatment of any of a variety of diseases, disorders,and/or conditions described herein.

Provided herein, are methods to prevent infection and/or sepsis in asubject at risk of developing infection and/or sepsis, the methodcomprising administering to a subject in need of such prevention acomposition comprising an oncology-related polynucleotide, primaryconstruct or mmRNA precursor encoding an anti-microbial polypeptide(e.g., an anti-bacterial polypeptide), or a partially or fully processedform thereof in an amount sufficient to prevent infection and/or sepsis.In certain embodiments, the subject at risk of developing infectionand/or sepsis may be a cancer patient. In certain embodiments, thecancer patient may have undergone a conditioning regimen. In someembodiments, the conditioning regiment may include, but is not limitedto, chemotherapy, radiation therapy, or both.

Further provided herein, are methods to treat infection and/or sepsis ina subject, the method comprising administering to a subject in need ofsuch treatment a composition comprising an oncology-relatedpolynucleotide, primary construct or mmRNA precursor encoding ananti-microbial polypeptide (e.g., an anti-bacterial polypeptide), e.g.,an anti-microbial polypeptide described herein, or a partially or fullyprocessed form thereof in an amount sufficient to treat an infectionand/or sepsis. In certain embodiments, the subject in need of treatmentis a cancer patient. In certain embodiments, the cancer patient hasundergone a conditioning regimen. In some embodiments, the conditioningregiment may include, but is not limited to, chemotherapy, radiationtherapy, or both.

In certain embodiments, the subject may exhibits acute or chronicmicrobial infections (e.g., bacterial infections). In certainembodiments, the subject may have received or may be receiving atherapy. In certain embodiments, the therapy may include, but is notlimited to, radiotherapy, chemotherapy, steroids, ultraviolet radiation,or a combination thereof. In certain embodiments, the patient may sufferfrom a microvascular disorder. In some embodiments, the microvasculardisorder may be diabetes. In certain embodiments, the patient may have awound. In some embodiments, the wound may be an ulcer. In a specificembodiment, the wound may be a diabetic foot ulcer. In certainembodiments, the subject may have one or more burn wounds. In certainembodiments, the administration may be local or systemic. In certainembodiments, the administration may be subcutaneous. In certainembodiments, the administration may be intravenous. In certainembodiments, the administration may be oral. In certain embodiments, theadministration may be topical. In certain embodiments, theadministration may be by inhalation. In certain embodiments, theadministration may be rectal. In certain embodiments, the administrationmay be vaginal.

Other aspects of the present disclosure relate to transplantation ofcells containing oncology-related polynucleotide, primary construct, ormmRNA to a mammalian subject. Administration of cells to mammaliansubjects is known to those of ordinary skill in the art, and include,but is not limited to, local implantation (e.g., topical or subcutaneousadministration), organ delivery or systemic injection (e.g., intravenousinjection or inhalation), and the formulation of cells inpharmaceutically acceptable carrier. Such compositions containingoncology-related polynucleotide, primary construct, or mmRNA can beformulated for administration intramuscularly, transarterially,intraperitoneally, intravenously, intranasally, subcutaneously,endoscopically, transdermally, or intrathecally. In some embodiments,the composition may be formulated for extended release.

The subject to whom the therapeutic agent may be administered suffersfrom or may be at risk of developing a disease, disorder, or deleteriouscondition. Provided are methods of identifying, diagnosing, andclassifying subjects on these bases, which may include clinicaldiagnosis, biomarker levels, genome-wide association studies (GWAS), andother methods known in the art.

Oncology-Related Applications

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs and/or oncology-related mmRNA may beused in the treatment, management, characterization and/or diagnosis ofcancer, a cancer-related and/or a cancer treatment-related disorder,side effect and/or condition. Such disease, disorders and conditionsinclude, but are not limited to, adrenal cortical cancer, advancedcancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer,bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer,childhood cancer, cancer of unknown primary origin, Castleman disease,cervical cancer, colon/rectal cancer, endometrial cancer, esophaguscancer, Ewing family of tumors, eye cancer, fallopian tube cancer,gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinalstromal tumors, gestational trophoblastic disease, Hodgkin disease,Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngealcancer, acute lymphocytic leukemia, acute myeloid leukemia, chroniclymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocyticleukemia, liver cancer, non-small cell lung cancer, small cell lungcancer, lung carcinoid tumor, lymphoma of the skin, malignantmesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer,osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer,pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma,salivary gland cancer, sarcoma in adult soft tissue, basal and squamouscell skin cancer, melanoma, small intestine cancer, stomach cancer,testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma,vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilmstumor.

In another embodiment, the oncology-related polynucleotides,oncology-related primary constructs and/or oncology-related mmRNA may beused in the treating, managing or manipulating at least onecancer-related or cancer treatment-related disorder, side effect orcondition such as chemo brain, peripheral neuropathy, fatigue,depression, nausea and vomiting, pain, anemia, lymphedema, infections,second cancers caused by cancer treatment, sexual side effects, reducedfertility or infertility, ostomies, insomnia and hair loss.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs and/or oncology-related mmRNA may beused to reduce the effect of at least one symptom of cancer in asubject. The symptom may include, but is not limited to, weakness, achesand pains, fever, fatigue, weight loss, blood clots, increased bloodcalcium levels, low white blood cell count, short of breath, dizziness,headaches, hyperpigmentation, jaundice, erthema, pruritis, excessivehair growth, change in bowel habits, change in bladder function,long-lasting sores, white patches inside the mouth, white spots on thetongue, unusual bleeding or discharge, thickening or lump on parts ofthe body, indigestion, trouble swallowing, changes in warts or moles,change in new skin and nagging cough or hoarseness.

Common Categories of Cancer Brain Cancer

Brain cancer is the growth of abnormal cells in the tissues of the brainusually related to the growth of malignant brain tumors. Brain tumorsgrow and press on the nearby areas of the brain which can stop that partof the brain from working the way it should. Brain cancer rarely spreadsinto other tissues outside of the brain. The grade of tumor, based onhow abnormal the cancer cells look under a microscope, may be used totell the difference between slow- and fast-growing tumors. Grade Itumors grow slowly, rarely spreads into nearby tissues, has cells thatlook like normal cells and the entire tumor may be removable by surgery.Grade II tumors also grow slowly but may spread into nearby tissue andmay recur. Grade III tumors grow quickly, is likely to spread intonearby tissue and the tumor cells look very different from normal cells.Grade IV, high-grade, grows and spreads very quickly and there may beareas of dead cells in the tumor. Symptoms of brain cancer may include,but are not limited to, morning headache or headache that goes awayafter vomiting, frequent nausea and vomiting, vision, hearing, andspeech problems, loss of balance and trouble walking, weakness on oneside of the body, unusual sleepiness or change in activity level,unusual changes in personality or behavior, seizures.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withbrain cancer by administering to said subject an isolated polynucleotideencoding an oncology-related polypeptide of interest. In one embodiment,the oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA of the present invention may beused to reduce, eliminate, or prevent tumor growth in a subject who hasbeen diagnosed or may be diagnosed with brain cancer by administering tosaid subject an isolated polynucleotide encoding an oncology-relatedpolypeptide of interest. In one embodiment, the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA of the present invention may be used to reduce and/or ameliorateat least one symptom of cancer in a subject who has been diagnosed ormay be diagnosed with brain cancer by administering to said subject anisolated polynucleotide encoding an oncology-related polypeptide ofinterest. In a further embodiment, the oncology-related polypeptide ofinterest may include, but not limited to, SEQ ID NOs: 4711, 4712, 4713,4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722, 4723, 4724, 4725,4726, 4727, 4728, 4742, 4743, 4744, 4745, 4746, 4747, 4748, 4749, 4750,4751, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4825,4826, 4827, 4828, 4829, 4830, 4832, 4854, 4855, 4856, 4857, 4858, 4859,4860, 4861, 4863, 4864, 4865, 4866, 4867, 4868, 4869, 4870, 4871, 4872,4873, 4885, 4886, 4887, 4888, 4889, 4890, 4891, 4892, 4893, 4894, 4911,4912, 4913, 4914, 4915, 4916, 4917, 4918, 4919, 4920, 4960, 4961, 4962,4963, 4964, 4965, 4966, 4967, 4968, 4969, 4970, 4971, 4972, 4973, 4974,4975, 4976, 4977, 4978, 4979, 4980, 4981, 4982, 4983, 4984, 4985, 4986,4987, 4988, 4989, 4990, 4991, 4992, 4993, 4994, 4995, 4996, 4997, 4998,4999, 5000, 5001, 5002, 5008, 5009, 5010, 5011, 5012, 5013, 5014, 5015,5016, 5017, 5018, 5019, 5020, 5021, 5022, 5023, 5024, 5025, 5026, 5027,5028, 5030, 5031, 5032, 5033, 5034, 5035, 5036, 5037, 5038, 5039, 5040,5041, 5042, 5043, 5044, 5045, 5046, 5047, 5048, 5049, 5050, 5051, 5052,5053, 5054, 5055, 5056, 5057, 5058, 5059, 5060, 5061, 5062, 5063, 5064,5065, 5066, 5067, 5068, 5069, 5070, 5071, 5072, 5073, 5074, 5075, 5076,5077, 5078, 5079, 5103, 5104, 5105, 5106, 5107, 5108, 5109, 5110, 5118,5119, 5120, 5121, 5122, 5123, 5124, 5125, 5126, 5127, 5128, 5129, 5130,5131, 5132, 5133, 5134, 5135, 5136, 5137, 5138, 5139, 5140, 5141, 5145,5146, 5147, 5148, 5149, 5150, 5151, 5152, 5153, 5154, 5155, 5156, 5157,5158, 5159, 5160, 5161, 5162, 5163, 5164, 5165, 5166, 5167, 5168, 5169,5170, 5171, 5172, 5173, 5174, 5175, 5176, 5177, 5178, 5182, 5183, 5184,5185, 5186, 5187, 5188, 5189, 5190, 5191, 5192, 5193, 5194, 5195, 5196,5197, 5198, 5199, 5200, 5201, 5202, 5203, 5204, 5205, 5206, 5207, 5208,5209, 5210, 5211, 5212, 5213, 5230, 5231, 5252, 5253, 5254, 5255, 5256,5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264, 5265, 5266, 5267, 5268,5269, 5270, 5271, 5291, 5292, 5293, 5294, 5295, 5296, 5297, 5298, 5299,5300, 5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311,5312, 5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320, 5321, 5322, 5323,5324, 5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332, 5333, 5334, 5335,5336, 5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344, 5345, 5346, 5347,5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358, 5359,5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370, 5371,5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380, 5381, 5382, 5383,5384, 5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392, 5393, 5394, 5395,5396, 5397, 5398, 5399, 5400, 5401, 5402, 5419, 5420, 5421, 5426, 5427,5428, 5429, 5438, 5447, 5448, 5449, 5450, 5451, 5452, 5453, 5454, 5455,5456, 5457, 5458, 5459, 5460, 5461, 5462, 5463, 5464, 5465, 5469, 5470,5477, 5478, 5479, 5480, 5481, 5482, 5490, 5491, 5492, 5493, 5494, 5495,5496, 5497, 5498, 5499, 5504, 5505, 5506, 5507, 5508, 5509, 5510, 5511,5512, 5513, 5514, 5515, 5516, 5517, 5518, 5519, 5520, 5521, 5522, 5523,5524, 5525, 5526, 5527, 5528, 5529, 5530, 5531, 5532, 5533, 5534, 5535,5536, 5548, 5549, 5551, 5552, 5553, 5554, 5555, 5556, 5557, 5558, 5559,5560, 5561, 5562, 5563, 5564, 5565, 5566, 5567, 5568, 5569, 5570, 5571,5577, 5578, 5579, 5580, 5590, 5591, 5592, 5593, 5594, 5595, 5596, 5605,5606, 5607, 5608, 5609, 5610, 5611, 5616, 5617, 5618, 5619, 5620, 5621,5622, 5623, 5631, 5632, 5633, 5638, 5639, 5640, 5671, 5672, 5673, 5674,5675, 5676, 5677, 5678, 5679, 5680, 5681, 5688, 5689, 5690, 5691, 5692,5693, 5694, 5695, 5696, 5697, 5698, 5699, 5700, 5701, 5702, 5703, 5708,5709, 5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717, 5727, 5740, 5741,5743, 5744, 5745, 5746, 5747, 5748, 5749, 5750, 5751, 5755, 5757, 5758,5759, 5760, 5761, 5762, 5763, 5764, 5765, 5766, 5767, 5768, 5769, 5770,5772, 5773, 5775, 5776, 5777, 5778, 5779, 5780, 5783, 5784, 5785, 5786,5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796, 5797, 5798,5799, 5800, 5801, 5802, 5803, 5811, 5812, 5813, 5814, 5821, 5822, 5823,5824, 5825, 5826, 5827, 5828, 5829, 5830, 5831, 5833, 5834, 5836, 5837,5838, 5839, 5848, 5849, 5850, 5851, 5852, 5853, 5854, 5855, 5864, 5865,5866, 5867, 5868, 5875, 5876, 5877, 5878, 5880, 5881, 5882, 5883, 5884,5885, 5886, 5887, 5888, 5889, 5890, 5891, 5892, 5893, 5894, 5895, 5896,5897, 5898, 5899, 5907, 5908, 5909, 5911, 5912, 5913, 5914, 5915, 5916,5917, 5918, 5919, 5920, 5921, 5922, 5929, 5950, 5951, 5952, 5953, 5954,5955, 5956, 5963, 5967, 5969, 5970, 5982, 5983, 5984, 5985, 5986, 5987,5988, 5997, 5998, 5999, 6000, 6001, 6002, 6068, 6069, 6070, 6071, 6072,6074, 6075, 6076, 6077, 6078, 6080, 6081, 6082, 6083, 6084, 6085, 6086,6087, 6088, 6089, 6090, 6091, 6092, 6093, 6094, 6095, 6096, 6097, 6098,6100, 6101, 6102, 6103, 6105, 6106, 6107, 6108, 6109, 6110, 6111, 6112,6113, 6114, 6115, 6116, 6117, 6118, 6119, 6120, 6121, 6122, 6123, 6124,6128, 6130, 6142, 6143, 6144, 6145, 6146, 6147, 6148, 6149, 6151, 6152,6153, 6154, 6155, 6156, 6157, 6169, 6170, 6171, 6172, 6173, 6174, 6175,6176, 6177, 6178, 6179, 6180, 6181, 6182, 6185, 6186, 6187, 6191, 6208,6209, 6231, 6232, 6233, 6234, 6235, 6236, 6237, 6238, 6239, 6251, 6256,6257, 6258, 6259, 6260, 6261, 6262, 6278, 6279, 6280, 6281, 6282, 6286,6287, 6288, 6289, 6290, 6298, 6299, 6300, 6301, 6302, 6303, 6304, 6305,6306, 6307, 6308, 6309, 6310, 6311, 6312, 6313, 6314, 6315, 6356, 6357,6358, 6378, 6379, 6387, 6388, 6389, 6390, 6391, 6392, 6393, 6394, 6395,6396, 6397, 6398, 6399, 6400, 6401, 6402, 6403, 6405, 6406, 6407, 6451,6452, 6453, 6497, 6498, 6499, 6500, 6501, 6502, 6503, 6509, 6510, 6511,6512, 6514, 6519, 6520, 6521, 6522, 6523, 6549, 6550, 6551, 6552, 6553,6554, 6555, 6556, 6557, 6558, 6588, 6589, 6599, 6600, 6601, 6602, 6603,6604, 6605, 6606, 6611, 6612, 6613, 6614, 6615, 6616, 6617, 6618, 6619,6620, 6621, 6622, 6623, 6624, 6625, 6626, 6627, 6628, 6629, 6630, 6631,6632, 6633, 6634, 6638, 6639, 6640, 6641, 6642, 6643, 6644, 6658, 6684,6685, 6686, 6697, 6698, 6699, 6700, 6701, 6702, 6703, 6704, 6705, 6706,6707, 6708, 6709, 6710, 6719, 6721, 6723, 6724, 6754, 6755, 6756, 6757,6758, 6759, 6760, 6761, 6762, 6763, 6764, 6796, 6797, 6798, 6799, 6800,6805, 6806, 6807, 6808, 6809, 6814, 6815, 6816, 6817, 6818, 6824, 6825,6826, 6831, 6832, 6833, 6834, 6853, 6854, 6855, 6856, 6857, 6860, 6861,6862, 6863, 6864, 6865, 6866, 6867, 6868, 6869, 6872, 6873, 6875, 6876,6877, 6878, 6879, 6885, 6888, 6889, 6890, 6891, 6892, 6893, 6894, 6895,6896, 6897, 6898, 6899, 6900, 6901, 6902, 6903, 6904, 6905, 6906, 6907,6908, 6909, 6910, 6911, 6912, 6913, 6914, 6915, 6916, 6917, 6918, 6919,6920, 6921, 6922, 6923, 6924, 6925, 6926, 6927, 6928, 6929, 6930, 6931,6932, 6933, 6934, 6935, 6936, 6953, 6954, 6955, 6957, 6958, 6959, 6960,6962, 6963, 6964, 6965, 6966, 6967, 6968, 6969, 6970, 6971, 6972, 6973,6983, 6984, 6985, 6988, 6989, 6990, 6991, 6992, 6993, 6994, 6995, 7007,7008, 7009, 7010, 7011, 7012, 7015, 7016, 7017, 7018, 7019, 7020, 7021,7034, 7035, 7084, 7086, 7087, 7088, 7089, 7090, 7091, 7092, 7128, 7137,7138, 7139, 7140, 7141, 7142, 7143, 7144, 7145, 7146, 7147, 7148, 7149,7150, 7151, 7152, 7153, 7154, 7155, 7195, 7196, 7197, 7198, 7200, 7201,7202, 7203, 7204, 7205, 7206, 7207, 7208, 7209, 7210, 7211, 7212, 7213,7214, 7215, 7216, 7217, 7218, 7219, 7220, 7221, 7222, 7223, 7224, 7225,7226, 7227, 7228, 7229, 7230, 7231, 7232, 7233, 7234, 7235, 7236, 7237,7238, 7239, 7240, 7241, 7243, 7244, 7245, 7246, 7247, 7248, 7249, 7250,7251, 7252, 7253, 7254, 7255, 7256, 7257, 7258, 7259, 7260, 7261, 7262,7263, 7264, 7265, 7266, 7267, 7268, 7269, 7270, 7271, 7291, 7292, 7293,7294, 7295, 7296, 7297, 7298, 7305, 7306, 7307, 7308, 7309, 7310, 7311,7312, 7313, 7314, 7315, 7316, 7317, 7347, 7348, 7349, 7358, 7359, 7360,7361, 7362, 7363, 7364, 7365, 7372, 7373, 7374, 7375, 7376, 7377, 7378,7383, 7415, 7416, 7417, 7438, 7471, 7472, 7473, 7474, 7475, 7476, 7477,7490, 7496, 7497, 7498, 7499, 7500, 7501, 7502, 7503, 7504, 7505, 7506,7507, 7508, 7509, 7510, 7511, 7512, 7513, 7514, 7515, 7532, 7533, 7534,7535, 7537, 7538, 7539, 7540, 7541, 7542, 7543, 7544, 7545, 7546, 7547,7548, 7549, 7550, 7551, 7552, 7553, 7554, 7555, 7556, 7557, 7558, 7559,7560, 7585, 7586, 7587, 7588, 7589, 7590, 7591, 7592, 7593, 7594, 7595,7596, 7597, 7599, 7600, 7601, 7617, 7618, 7619, 7620, 7621, 7622, 7623,7638, 7668, 7669, 7670, 7672, 7673, 7674, 7675, 7676, 7685, 7687, 7694,7695, 7696, 7717, 7718, 7719, 7720, 7721, 7722, 7723, 7724, 7725, 7726,7727, 7728, 7729, 7730, 7731, 7732, 7733, 7734, 7735, 7736, 7737, 7738,7739, 7740, 7741, 7742, 7743, 7744, 7746, 7747, 7748, 7749, 7750, 7751,7752, 7757, 7758, 7759, 7760, 7761, 7762, 7763, 7764, 7765, 7766, 7767,7768, 7796, 7797, 7805, 7813, 7814, 7815, 7816, 7817, 7818, 7819, 7820,7821, 7822, 7848, 7849, 7850, 7851, 7852, 7853, 7854, 7859, 7860, 7861,7862, 7863, 7864, 7865, 7866, 7867, 7869, 7870, 7874, 7875, 7917, 7918,7919, 7925, 7926, 7927, 7928, 7929, 7941, 7942, 7943, 7944, 7945, 7946,7947, 7948, 7949, 7950, 7951, 7952, 7953, 7955, 7956, 7957, 7958, 7959,7960, 7961, 7962, 7963, 7964, 7965, 7966, 7967, 7968, 7969, 7970, 7971,7972, 7974, 7975, 7976, 7977, 7978, 7979, 7980, 7981, 7982, 7983, 7984,7985, 7986, 7987, 7988, 7989, 7990, 7991, 7992, 7993, 7994, 7995, 7996,7997, 7998, 7999, 8000, 8011, 8012, 8013, 8014, 8015, 8016, 8017, 8018,8019, 8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027, 8028, 8029, 8030,8048, 8049, 8050, 8051, 8052, 8053, 8054, 8055, 8056, 8057, 8087, 8088,8089, 8090, 8091, 8092, 8095, 8096, 8097, 8098, 8099, 8100, 8101, 8102,8103, 8104, 8105, 8106, 8107, 8110, 8111, 8112, 8113, 8114, 8115, 8116,8117, 8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125, 8126, 8127, 8130,8131, 8132, 8133, 8134, 8135, 8136, 8144, 8145, 8166, 8169, 8170, 8171,8172, 8173, 8174, 8175, 8176, 8177, 8178, 8179, 8180, 8181, 8182, 8188,8191, 8192, 8193, 8194, 8195, 8196, 8197, 8198, 8199, 8200, 8201, 8209,8210, 8211, 8212, 8215, 8227, 8230, 8231, 8232, 8233, 8234, 8235, 8236,8237, 8238, 8239, 8240, 8241, 8242, 8243, 8244, 8245, 8246, 8247, 8248,8249, 8250, 8251, 8260, 8261, 8262, 8263, 8299, 8302, 8303, 8304, 8305,8306, 8353, 8354, 8355, 8356, 8357, 8358, 8359, 8360, 8361, 8375, 8376,8377, 8378, 8379, 8380, 8381, 8382, 8410, 8411, 8412, 8440, 8459, 8460,8461, 8462, 8463, 8474, 8475, 8476, 8477, 8478, 8508, 8509, 8510, 8511,8512, 8514, 8515, 8546, 8547, 8548, 8549, 8550, 8551, 8552, 8553, 8554,8555, 8570, 8571, 8572, 8573, 8574, 8575, 8576, 8577, 8578, 8579, 8580,8581, 8586, 8587, 8593, 8594, 8595, 8596, 8597, 8601, 8602, 8603, 8604,8605, 8615, 8617, 8618, 8655, 8686, 8709, 8712, 8713, 8714, 8715, 8716,8717, 8718, 8719, 8720, 8721, 8722, 8723, 8724, 8725, 8726, 8727, 8728,8729, 8730, 8731, 8732, 8733, 8734, 8770, 8771, 8788, 8789, 8790, 8791,8792, 8793, 8794, 8795, 8796, 8797, 8798, 8799, 8800, 8801, 8802, 8803,8804, 8805, 8806, 8810, 8811, 8817, 8818, 8819, 8820, 8821, 8822, 8823,8829, 8830, 8837, 8838, 8839, 8840, 8841, 8848, 8849, 8850, 8851, 8854,8855, 8856, 8857, 8858, 8859, 8860, 8861, 8862, 8863, 8864, 8865, 8867,8868, 8872, 8873, 8874, 8875, 8876, 8878, 8879, 8880, 8881, 8882, 8883,8884, 8885, 8886, 8887, 8888, 8889, 8890, 8891, 8892, 8893, 8894, 8895,8896, 8897, 8898, 8899, 8900, 8901, 8902, 8903, 8904, 8906, 8913, 8914,8915, 8916, 8917, 8918, 8919, 8920, 8921, 8925, 8926, 8927, 8928, 8933,8934, 8935, 8936, 8937, 8938, 8939, 8940, 8941, 8942, 8943, 8944, 8945,8946, 8947, 8948, 8949, 8950, 8951, 8954, 8955, 8956, 8992, 8993, 8994,8995, 8996, 8997, 8998, 8999, 9000, 9001, 9002, 9003, 9010, 9011, 9012,9013, 9014, 9018, 9019, 9020, 9021, 9022, 9023, 9024, 9025, 9026, 9027,9028, 9029, 9030, 9031, 9041, 9042, 9043, 9044, 9045, 9046, 9047, 9048,9049, 9050, 9051, 9052, 9053, 9054, 9055, 9056, 9057, 9058, 9059, 9065,9066, 9087, 9088, 9091, 9092, 9093, 9094, 9095, 9096, 9097, 9098, 9103,9104, 9105, 9106, 9107, 9108, 9109, 9110, 9123, 9124, 9125, 9126, 9127,9128, 9129, 9130, 9131, 9133, 9134, 9135, 9136, 9137, 9145, 9146, 9147,9148, 9149, 9150, 9151, 9160, 9161, 9162, 9163, 9201, 9203.

Breast Cancer

Breast cancer forms in the tissues of the breast, of both men and women,such as, but not limited to, the ducts and the lobules. The most commontype of breast cancer is ductal carcinoma which begins in the cells ofthe ducts. Lobular cancer, which begins in the lobes or lobules, isoften found in both breasts. An uncommon type of breast cancer,inflammatory breast cancer, causes the breast to be warm, red andswollen. Hereditary breast cancer makes up approximately 5-10% of allbreast cancer and altered genes are common in some ethnic groups makingthat ethnic group more susceptible to breast cancer. Symptoms of breastcancer include, but are not limited to, a lump or thickening in or nearthe breast or in the underarm area, change in the size or shape of thebreast, dimple or puckering in the skin of the breast, inward turnednipple of the breast, fluid from the nipple which is not breast milk,scaly, red or swollen skin on the breast, nipple, or areola, and dimplesin the breast that look like the skin of orange (peau d'orange).

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withbreast cancer by administering to said subject an isolatedpolynucleotide encoding an oncology-related polypeptide of interest. Inone embodiment, the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA of the present inventionmay be used to reduce, eliminate, or prevent tumor growth in a subjectwho has been diagnosed or may be diagnosed with breast cancer byadministering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In one embodiment, theoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA of the present invention may be used to reduceand/or ameliorate at least one symptom of cancer in a subject who hasbeen diagnosed or may be diagnosed with breast cancer by administeringto said subject an isolated polynucleotide encoding an oncology-relatedpolypeptide of interest. In a further embodiment, the oncology-relatedpolypeptide of interest may include, but not limited to, SEQ ID NOs:4711, 4712, 4713, 4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722,4723, 4724, 4725, 4726, 4727, 4728, 4742, 4743, 4744, 4745, 4746, 4747,4748, 4749, 4750, 4751, 4753, 4756, 4757, 4758, 4759, 4761, 4762, 4776,4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788,4789, 4790, 4825, 4826, 4827, 4828, 4829, 4830, 4832, 4835, 4836, 4854,4855, 4856, 4857, 4858, 4859, 4860, 4861, 4863, 4864, 4865, 4866, 4867,4868, 4869, 4870, 4871, 4872, 4873, 4885, 4886, 4887, 4888, 4889, 4890,4891, 4892, 4893, 4894, 4911, 4912, 4913, 4914, 4915, 4916, 4917, 4918,4919, 4920, 4946, 4947, 4948, 4949, 4950, 4958, 4959, 4960, 4961, 4962,4963, 4964, 4965, 4966, 4967, 4968, 4969, 4970, 4971, 4972, 4973, 4974,4975, 4976, 4977, 4978, 4979, 4980, 4981, 4982, 4983, 4984, 4985, 4986,4987, 4988, 4989, 4990, 4991, 4992, 4993, 4994, 4995, 4996, 4997, 4998,4999, 5000, 5001, 5002, 5008, 5009, 5010, 5011, 5012, 5013, 5014, 5015,5016, 5017, 5018, 5019, 5020, 5021, 5022, 5023, 5024, 5025, 5026, 5027,5028, 5030, 5031, 5032, 5033, 5034, 5035, 5036, 5037, 5038, 5039, 5040,5041, 5042, 5043, 5044, 5045, 5046, 5047, 5048, 5049, 5050, 5051, 5052,5053, 5054, 5055, 5056, 5057, 5058, 5059, 5060, 5061, 5062, 5063, 5064,5065, 5066, 5067, 5068, 5069, 5070, 5071, 5072, 5073, 5074, 5075, 5076,5077, 5078, 5079, 5103, 5104, 5105, 5106, 5107, 5108, 5109, 5110, 5118,5119, 5120, 5121, 5122, 5123, 5124, 5125, 5126, 5127, 5128, 5129, 5130,5131, 5132, 5133, 5134, 5135, 5136, 5137, 5138, 5139, 5140, 5141, 5142,5143, 5144, 5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152, 5153, 5154,5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164, 5165, 5166,5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176, 5177, 5178,5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191, 5192, 5193,5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203, 5204, 5205,5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5214, 5215, 5216, 5217,5218, 5219, 5220, 5221, 5222, 5223, 5230, 5231, 5252, 5253, 5254, 5255,5256, 5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264, 5265, 5266, 5267,5268, 5269, 5270, 5271, 5282, 5291, 5292, 5293, 5294, 5295, 5296, 5297,5298, 5299, 5300, 5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308, 5309,5310, 5311, 5312, 5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320, 5321,5322, 5323, 5324, 5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332, 5333,5334, 5335, 5336, 5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344, 5345,5346, 5347, 5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357,5358, 5359, 5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369,5370, 5371, 5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380, 5381,5382, 5383, 5384, 5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392, 5393,5419, 5420, 5421, 5426, 5427, 5428, 5429, 5438, 5447, 5448, 5449, 5450,5451, 5452, 5453, 5454, 5455, 5456, 5457, 5458, 5459, 5460, 5461, 5462,5463, 5464, 5465, 5469, 5470, 5477, 5478, 5479, 5480, 5481, 5482, 5490,5491, 5492, 5493, 5494, 5495, 5496, 5497, 5498, 5499, 5504, 5505, 5506,5507, 5508, 5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516, 5517, 5518,5519, 5520, 5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529, 5530,5531, 5532, 5533, 5534, 5535, 5536, 5548, 5549, 5551, 5552, 5553, 5554,5555, 5556, 5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565, 5566,5567, 5568, 5569, 5570, 5571, 5577, 5578, 5579, 5580, 5590, 5591, 5592,5593, 5594, 5595, 5596, 5605, 5606, 5607, 5608, 5609, 5610, 5611, 5616,5617, 5618, 5619, 5620, 5621, 5622, 5623, 5631, 5632, 5633, 5638, 5639,5640, 5660, 5661, 5662, 5663, 5664, 5665, 5671, 5672, 5673, 5674, 5675,5676, 5677, 5678, 5679, 5680, 5681, 5688, 5689, 5690, 5691, 5692, 5693,5694, 5695, 5696, 5697, 5698, 5699, 5700, 5701, 5702, 5703, 5704, 5705,5706, 5707, 5708, 5709, 5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717,5727, 5740, 5741, 5742, 5743, 5744, 5745, 5746, 5747, 5748, 5749, 5750,5751, 5752, 5753, 5754, 5755, 5757, 5758, 5759, 5760, 5761, 5762, 5763,5764, 5765, 5766, 5767, 5768, 5769, 5770, 5772, 5773, 5775, 5776, 5777,5778, 5779, 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788, 5789,5790, 5791, 5792, 5793, 5794, 5795, 5796, 5797, 5798, 5799, 5800, 5801,5802, 5803, 5806, 5807, 5808, 5809, 5810, 5811, 5812, 5813, 5814, 5821,5822, 5823, 5824, 5825, 5826, 5827, 5828, 5829, 5830, 5831, 5832, 5833,5834, 5836, 5837, 5838, 5839, 5848, 5849, 5850, 5851, 5852, 5853, 5854,5855, 5864, 5865, 5866, 5867, 5868, 5875, 5876, 5877, 5878, 5880, 5881,5882, 5883, 5884, 5885, 5886, 5887, 5888, 5889, 5890, 5891, 5892, 5893,5894, 5895, 5896, 5897, 5898, 5899, 5907, 5908, 5909, 5911, 5912, 5913,5914, 5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922, 5929, 5950, 5951,5952, 5953, 5954, 5955, 5956, 5963, 5967, 5969, 5970, 5982, 5983, 5984,5985, 5986, 5987, 5988, 5997, 5998, 5999, 6000, 6001, 6002, 6068, 6069,6070, 6071, 6072, 6074, 6075, 6076, 6077, 6078, 6080, 6081, 6082, 6083,6084, 6085, 6086, 6087, 6088, 6089, 6090, 6091, 6092, 6093, 6094, 6095,6096, 6097, 6098, 6100, 6101, 6102, 6103, 6105, 6106, 6107, 6108, 6109,6110, 6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118, 6119, 6120, 6121,6122, 6123, 6124, 6128, 6130, 6142, 6143, 6144, 6145, 6146, 6147, 6148,6149, 6151, 6152, 6153, 6154, 6155, 6156, 6157, 6169, 6170, 6171, 6172,6173, 6174, 6175, 6176, 6177, 6178, 6179, 6180, 6181, 6182, 6185, 6186,6187, 6191, 6208, 6209, 6231, 6232, 6233, 6234, 6235, 6236, 6237, 6238,6239, 6251, 6256, 6257, 6258, 6259, 6260, 6261, 6262, 6278, 6279, 6280,6281, 6282, 6283, 6284, 6286, 6287, 6288, 6289, 6290, 6298, 6299, 6300,6301, 6302, 6303, 6304, 6305, 6306, 6307, 6308, 6309, 6310, 6311, 6312,6313, 6314, 6315, 6327, 6328, 6329, 6333, 6334, 6335, 6336, 6337, 6338,6339, 6340, 6356, 6357, 6358, 6378, 6379, 6387, 6388, 6389, 6390, 6391,6392, 6393, 6394, 6395, 6396, 6397, 6398, 6399, 6400, 6401, 6402, 6403,6405, 6406, 6407, 6451, 6452, 6453, 6497, 6498, 6499, 6500, 6501, 6502,6503, 6509, 6510, 6511, 6512, 6514, 6519, 6520, 6521, 6522, 6523, 6524,6525, 6549, 6550, 6551, 6552, 6553, 6554, 6555, 6556, 6557, 6558, 6588,6589, 6599, 6600, 6601, 6602, 6603, 6604, 6605, 6606, 6611, 6612, 6613,6614, 6615, 6616, 6617, 6618, 6619, 6620, 6621, 6622, 6623, 6624, 6625,6626, 6627, 6628, 6629, 6630, 6631, 6632, 6633, 6634, 6638, 6639, 6640,6641, 6642, 6643, 6644, 6684, 6685, 6686, 6697, 6698, 6699, 6700, 6701,6718, 6719, 6721, 6723, 6724, 6737, 6738, 6739, 6740, 6741, 6742, 6743,6747, 6748, 6749, 6750, 6751, 6752, 6754, 6755, 6756, 6757, 6758, 6759,6760, 6761, 6762, 6763, 6764, 6796, 6797, 6798, 6799, 6800, 6805, 6806,6807, 6808, 6809, 6814, 6815, 6816, 6817, 6818, 6824, 6825, 6826, 6831,6832, 6833, 6834, 6853, 6854, 6855, 6856, 6857, 6860, 6861, 6862, 6863,6864, 6865, 6866, 6867, 6868, 6869, 6870, 6871, 6872, 6873, 6875, 6876,6877, 6878, 6879, 6885, 6888, 6889, 6890, 6891, 6892, 6893, 6894, 6895,6896, 6897, 6898, 6899, 6900, 6901, 6902, 6903, 6904, 6905, 6906, 6907,6908, 6909, 6910, 6911, 6912, 6913, 6914, 6915, 6916, 6917, 6918, 6919,6920, 6921, 6922, 6923, 6924, 6925, 6926, 6927, 6928, 6929, 6930, 6931,6932, 6933, 6934, 6935, 6936, 6953, 6954, 6955, 6957, 6958, 6959, 6960,6962, 6963, 6964, 6965, 6966, 6967, 6968, 6969, 6970, 6971, 6972, 6973,6983, 6984, 6985, 6988, 6989, 6990, 6991, 6992, 6993, 6994, 6995, 7007,7008, 7009, 7010, 7011, 7012, 7015, 7016, 7017, 7018, 7019, 7020, 7021,7027, 7028, 7029, 7030, 7034, 7035, 7045, 7046, 7047, 7048, 7049, 7050,7051, 7052, 7053, 7054, 7055, 7056, 7057, 7058, 7059, 7060, 7084, 7086,7087, 7088, 7089, 7090, 7091, 7092, 7093, 7094, 7095, 7096, 7097, 7098,7099, 7100, 7101, 7102, 7103, 7104, 7105, 7106, 7125, 7126, 7127, 7128,7129, 7130, 7131, 7132, 7133, 7134, 7135, 7136, 7137, 7138, 7139, 7140,7141, 7142, 7143, 7144, 7145, 7146, 7147, 7148, 7149, 7150, 7151, 7152,7153, 7154, 7155, 7195, 7196, 7197, 7198, 7200, 7201, 7202, 7203, 7204,7205, 7206, 7207, 7208, 7209, 7210, 7211, 7212, 7213, 7214, 7215, 7216,7217, 7218, 7219, 7220, 7221, 7222, 7223, 7224, 7225, 7226, 7227, 7228,7229, 7230, 7231, 7232, 7233, 7234, 7235, 7236, 7237, 7238, 7239, 7240,7241, 7243, 7244, 7245, 7246, 7247, 7248, 7249, 7250, 7251, 7252, 7253,7254, 7255, 7256, 7257, 7258, 7259, 7260, 7261, 7262, 7263, 7264, 7265,7266, 7267, 7268, 7269, 7270, 7271, 7291, 7292, 7293, 7294, 7295, 7296,7297, 7298, 7347, 7348, 7349, 7358, 7359, 7360, 7361, 7362, 7363, 7364,7365, 7368, 7369, 7370, 7371, 7372, 7373, 7374, 7375, 7376, 7377, 7378,7380, 7383, 7415, 7416, 7417, 7438, 7443, 7444, 7445, 7446, 7447, 7448,7449, 7450, 7451, 7452, 7453, 7454, 7455, 7456, 7457, 7471, 7472, 7473,7474, 7475, 7476, 7477, 7484, 7490, 7496, 7497, 7498, 7499, 7500, 7501,7502, 7503, 7504, 7505, 7506, 7507, 7508, 7509, 7510, 7511, 7512, 7513,7514, 7515, 7532, 7533, 7534, 7535, 7537, 7538, 7539, 7540, 7541, 7542,7543, 7544, 7545, 7546, 7547, 7548, 7549, 7550, 7551, 7552, 7553, 7554,7555, 7556, 7557, 7558, 7559, 7560, 7585, 7586, 7587, 7588, 7589, 7590,7591, 7592, 7593, 7595, 7596, 7597, 7599, 7600, 7601, 7617, 7618, 7619,7620, 7621, 7622, 7623, 7638, 7668, 7669, 7670, 7672, 7673, 7674, 7675,7676, 7685, 7687, 7694, 7695, 7696, 7717, 7718, 7719, 7720, 7721, 7722,7723, 7724, 7725, 7726, 7727, 7728, 7729, 7730, 7731, 7732, 7733, 7734,7735, 7736, 7737, 7738, 7739, 7740, 7741, 7742, 7743, 7744, 7746, 7747,7748, 7749, 7750, 7751, 7752, 7757, 7758, 7759, 7760, 7761, 7762, 7763,7764, 7765, 7766, 7767, 7768, 7796, 7797, 7798, 7799, 7800, 7801, 7802,7803, 7804, 7805, 7813, 7814, 7815, 7816, 7817, 7818, 7819, 7820, 7821,7822, 7848, 7849, 7850, 7851, 7852, 7853, 7854, 7859, 7860, 7861, 7862,7863, 7864, 7865, 7866, 7867, 7869, 7870, 7874, 7875, 7917, 7918, 7919,7925, 7926, 7927, 7928, 7929, 7941, 7942, 7943, 7944, 7945, 7946, 7947,7948, 7949, 7950, 7951, 7952, 7953, 7955, 7956, 7957, 7958, 7959, 7960,7961, 7962, 7963, 7964, 7965, 7966, 7967, 7968, 7969, 7970, 7971, 7972,7974, 7975, 7976, 7977, 7978, 7979, 7980, 7981, 7982, 7983, 7984, 7985,7986, 7987, 7988, 7989, 7990, 7991, 7992, 7993, 7994, 7995, 7996, 7997,7998, 7999, 8000, 8011, 8012, 8013, 8014, 8015, 8016, 8017, 8018, 8019,8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027, 8028, 8029, 8030, 8048,8049, 8050, 8051, 8052, 8053, 8054, 8055, 8056, 8057, 8087, 8088, 8089,8090, 8091, 8092, 8095, 8096, 8097, 8098, 8099, 8100, 8101, 8102, 8103,8104, 8105, 8106, 8107, 8110, 8111, 8112, 8113, 8114, 8115, 8116, 8117,8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125, 8126, 8127, 8130, 8131,8132, 8133, 8134, 8135, 8136, 8144, 8145, 8166, 8169, 8170, 8171, 8172,8173, 8174, 8175, 8176, 8177, 8178, 8179, 8180, 8181, 8182, 8183, 8184,8185, 8186, 8187, 8188, 8191, 8192, 8193, 8194, 8195, 8196, 8197, 8198,8199, 8200, 8201, 8209, 8210, 8211, 8212, 8215, 8227, 8230, 8231, 8232,8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241, 8242, 8243, 8244,8245, 8246, 8247, 8248, 8249, 8250, 8251, 8260, 8261, 8262, 8263, 8299,8302, 8303, 8304, 8305, 8306, 8313, 8314, 8353, 8354, 8355, 8356, 8357,8358, 8359, 8360, 8361, 8375, 8376, 8377, 8378, 8379, 8380, 8381, 8382,8410, 8411, 8412, 8440, 8459, 8460, 8461, 8462, 8463, 8474, 8475, 8476,8477, 8478, 8504, 8505, 8506, 8508, 8509, 8510, 8511, 8512, 8514, 8515,8546, 8547, 8548, 8549, 8550, 8551, 8552, 8553, 8554, 8555, 8568, 8569,8570, 8571, 8572, 8573, 8574, 8575, 8576, 8577, 8578, 8579, 8580, 8581,8586, 8587, 8593, 8594, 8595, 8596, 8597, 8601, 8602, 8603, 8604, 8605,8615, 8617, 8618, 8655, 8686, 8709, 8712, 8713, 8714, 8715, 8716, 8717,8718, 8719, 8720, 8721, 8722, 8723, 8724, 8725, 8726, 8727, 8728, 8729,8730, 8731, 8732, 8733, 8734, 8770, 8771, 8788, 8789, 8790, 8791, 8792,8793, 8794, 8795, 8796, 8797, 8798, 8799, 8800, 8801, 8802, 8803, 8804,8805, 8806, 8810, 8811, 8817, 8818, 8819, 8820, 8821, 8822, 8823, 8829,8830, 8835, 8836, 8837, 8838, 8839, 8840, 8841, 8848, 8849, 8850, 8851,8854, 8855, 8856, 8857, 8858, 8859, 8860, 8861, 8862, 8863, 8864, 8865,8867, 8868, 8872, 8873, 8874, 8875, 8876, 8878, 8879, 8880, 8881, 8882,8883, 8884, 8885, 8886, 8887, 8888, 8889, 8890, 8891, 8892, 8893, 8894,8895, 8896, 8897, 8898, 8899, 8900, 8901, 8902, 8903, 8904, 8905, 8906,8913, 8914, 8915, 8916, 8917, 8918, 8919, 8920, 8921, 8925, 8926, 8927,8928, 8933, 8934, 8935, 8936, 8937, 8938, 8939, 8940, 8941, 8942, 8943,8944, 8945, 8946, 8947, 8948, 8949, 8950, 8951, 8954, 8955, 8956, 8992,8993, 8994, 8995, 8996, 8997, 8998, 8999, 9000, 9001, 9002, 9003, 9010,9011, 9012, 9013, 9014, 9018, 9019, 9020, 9021, 9022, 9023, 9024, 9025,9026, 9027, 9028, 9029, 9030, 9031, 9036, 9037, 9038, 9039, 9040, 9041,9042, 9043, 9044, 9045, 9046, 9047, 9048, 9049, 9050, 9051, 9052, 9053,9054, 9055, 9056, 9057, 9058, 9059, 9065, 9066, 9087, 9088, 9091, 9092,9093, 9094, 9095, 9096, 9097, 9098, 9099, 9100, 9101, 9102, 9103, 9104,9105, 9106, 9107, 9108, 9109, 9110, 9123, 9124, 9125, 9126, 9127, 9128,9129, 9130, 9131, 9133, 9134, 9135, 9136, 9137, 9145, 9146, 9147, 9148,9149, 9150, 9151, 9160, 9161, 9162, 9163, 9201, 9203.

Cervical Cancer

Cervical cancer forms in the tissues of the cervix and is usuallyslow-growing. The cause of cervical cancer usually related to the humanpapillomavirus (HPV) infection. Although cervical cancer may not notshow any signs, possible symptoms may include, but are not limited to,vaginal bleeding, unusual vaginal discharge, pelvic pain and pain duringsexual intercourse.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withcervical cancer by administering to said subject an isolatedpolynucleotide encoding an oncology-related polypeptide of interest. Inone embodiment, the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA of the present inventionmay be used to reduce, eliminate, or prevent tumor growth in a subjectwho has been diagnosed or may be diagnosed with cervical cancer byadministering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In one embodiment, theoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA of the present invention may be used to reduceand/or ameliorate at least one symptom of cancer in a subject who hasbeen diagnosed or may be diagnosed with cervical cancer by administeringto said subject an isolated polynucleotide encoding an oncology-relatedpolypeptide of interest. In a further embodiment, the oncology-relatedpolypeptide of interest may include, but not limited to, SEQ ID NOs:4711, 4712, 4713, 4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722,4723, 4724, 4725, 4726, 4727, 4728, 4742, 4743, 4744, 4745, 4746, 4747,4748, 4749, 4750, 4751, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788,4789, 4790, 4825, 4826, 4827, 4828, 4829, 4830, 4832, 4854, 4855, 4856,4857, 4858, 4859, 4860, 4861, 4863, 4864, 4865, 4866, 4867, 4868, 4869,4871, 4873, 4885, 4886, 4887, 4888, 4889, 4890, 4891, 4892, 4893, 4894,4911, 4912, 4913, 4914, 4915, 4916, 4917, 4918, 4919, 4920, 4960, 4961,4962, 4964, 4965, 4966, 4967, 4968, 4969, 4970, 4971, 4972, 4973, 4974,4975, 4978, 4979, 4980, 4981, 4982, 4983, 4984, 4985, 4986, 4987, 4988,4989, 4990, 4991, 4992, 4993, 4994, 4995, 4996, 4997, 4998, 4999, 5000,5001, 5002, 5008, 5009, 5010, 5011, 5012, 5013, 5014, 5015, 5016, 5017,5018, 5019, 5020, 5021, 5022, 5023, 5024, 5025, 5026, 5027, 5028, 5030,5031, 5032, 5033, 5034, 5035, 5036, 5037, 5038, 5039, 5040, 5041, 5042,5043, 5044, 5045, 5046, 5047, 5048, 5049, 5050, 5051, 5052, 5053, 5054,5055, 5056, 5060, 5061, 5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069,5070, 5071, 5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079, 5103, 5104,5105, 5106, 5107, 5108, 5109, 5110, 5118, 5119, 5120, 5121, 5122, 5123,5124, 5125, 5126, 5127, 5128, 5129, 5130, 5131, 5132, 5133, 5134, 5135,5136, 5137, 5138, 5139, 5140, 5141, 5146, 5147, 5148, 5149, 5150, 5151,5152, 5153, 5154, 5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163,5164, 5165, 5166, 5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175,5176, 5177, 5178, 5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190,5191, 5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202,5203, 5204, 5205, 5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5230,5231, 5252, 5253, 5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262,5263, 5264, 5265, 5266, 5267, 5268, 5269, 5270, 5271, 5291, 5292, 5293,5294, 5295, 5296, 5297, 5298, 5299, 5300, 5301, 5302, 5303, 5304, 5305,5306, 5307, 5308, 5309, 5310, 5311, 5316, 5317, 5318, 5319, 5320, 5321,5322, 5323, 5324, 5325, 5326, 5327, 5330, 5331, 5332, 5333, 5334, 5335,5336, 5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344, 5345, 5346, 5347,5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358, 5359,5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370, 5371,5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380, 5381, 5382, 5383,5384, 5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392, 5393, 5419, 5420,5421, 5426, 5427, 5428, 5429, 5438, 5447, 5448, 5449, 5450, 5451, 5452,5453, 5454, 5455, 5456, 5457, 5458, 5459, 5460, 5461, 5462, 5463, 5464,5465, 5469, 5470, 5477, 5478, 5479, 5480, 5481, 5482, 5490, 5491, 5492,5493, 5494, 5495, 5496, 5497, 5498, 5499, 5504, 5505, 5506, 5507, 5508,5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516, 5517, 5518, 5519, 5520,5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529, 5530, 5531, 5532,5533, 5534, 5535, 5536, 5548, 5549, 5551, 5552, 5553, 5554, 5555, 5556,5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565, 5566, 5567, 5568,5569, 5570, 5571, 5577, 5578, 5579, 5580, 5590, 5591, 5592, 5593, 5594,5595, 5596, 5605, 5606, 5607, 5608, 5609, 5610, 5611, 5616, 5617, 5618,5619, 5620, 5621, 5622, 5623, 5631, 5632, 5633, 5638, 5639, 5640, 5671,5672, 5673, 5674, 5675, 5676, 5677, 5678, 5679, 5680, 5681, 5688, 5689,5690, 5691, 5692, 5693, 5694, 5695, 5696, 5697, 5698, 5699, 5700, 5701,5702, 5703, 5708, 5709, 5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717,5727, 5740, 5741, 5743, 5744, 5745, 5746, 5747, 5748, 5749, 5750, 5751,5755, 5757, 5758, 5759, 5760, 5761, 5762, 5763, 5764, 5765, 5766, 5767,5768, 5769, 5770, 5772, 5773, 5775, 5776, 5777, 5778, 5779, 5780, 5783,5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795,5796, 5797, 5798, 5799, 5800, 5801, 5802, 5803, 5811, 5812, 5813, 5814,5821, 5822, 5823, 5824, 5825, 5826, 5827, 5828, 5829, 5830, 5831, 5833,5834, 5836, 5837, 5838, 5839, 5848, 5849, 5850, 5851, 5852, 5853, 5854,5855, 5864, 5865, 5866, 5867, 5868, 5875, 5876, 5877, 5878, 5880, 5881,5882, 5883, 5884, 5885, 5886, 5887, 5888, 5889, 5890, 5891, 5892, 5893,5894, 5895, 5896, 5897, 5898, 5899, 5907, 5908, 5909, 5911, 5912, 5913,5914, 5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922, 5929, 5950, 5951,5952, 5953, 5954, 5955, 5956, 5963, 5967, 5969, 5970, 5982, 5983, 5984,5985, 5986, 5987, 5988, 5997, 5998, 5999, 6000, 6001, 6002, 6068, 6069,6070, 6071, 6072, 6074, 6075, 6076, 6077, 6078, 6080, 6081, 6082, 6083,6084, 6085, 6086, 6087, 6088, 6089, 6090, 6091, 6092, 6093, 6094, 6095,6096, 6097, 6098, 6100, 6101, 6102, 6103, 6105, 6106, 6107, 6108, 6109,6110, 6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118, 6119, 6120, 6121,6122, 6123, 6124, 6128, 6130, 6142, 6143, 6144, 6145, 6146, 6147, 6148,6149, 6151, 6152, 6153, 6154, 6155, 6156, 6157, 6169, 6170, 6171, 6172,6173, 6174, 6175, 6176, 6177, 6178, 6179, 6180, 6181, 6182, 6185, 6186,6187, 6191, 6208, 6209, 6231, 6232, 6233, 6234, 6235, 6236, 6237, 6238,6239, 6251, 6256, 6257, 6258, 6259, 6260, 6261, 6262, 6278, 6279, 6280,6281, 6282, 6286, 6287, 6288, 6289, 6290, 6298, 6299, 6300, 6301, 6302,6303, 6304, 6305, 6306, 6307, 6308, 6309, 6310, 6311, 6312, 6313, 6314,6315, 6356, 6357, 6358, 6378, 6379, 6387, 6388, 6389, 6390, 6391, 6392,6393, 6394, 6395, 6396, 6397, 6398, 6399, 6400, 6401, 6402, 6403, 6405,6406, 6407, 6451, 6452, 6453, 6497, 6498, 6499, 6500, 6501, 6502, 6503,6509, 6510, 6511, 6512, 6514, 6519, 6520, 6521, 6522, 6523, 6550, 6551,6552, 6553, 6554, 6555, 6556, 6557, 6558, 6588, 6589, 6599, 6600, 6601,6602, 6603, 6604, 6605, 6606, 6611, 6612, 6613, 6614, 6615, 6616, 6617,6618, 6619, 6620, 6621, 6622, 6623, 6624, 6625, 6626, 6627, 6628, 6629,6630, 6631, 6632, 6633, 6634, 6638, 6639, 6640, 6641, 6642, 6643, 6644,6684, 6685, 6686, 6697, 6698, 6699, 6700, 6701, 6719, 6721, 6723, 6724,6754, 6755, 6756, 6757, 6758, 6759, 6760, 6761, 6762, 6763, 6764, 6796,6797, 6798, 6799, 6800, 6805, 6806, 6807, 6808, 6809, 6814, 6815, 6816,6817, 6818, 6824, 6825, 6826, 6831, 6832, 6833, 6834, 6853, 6854, 6855,6856, 6857, 6860, 6861, 6862, 6863, 6864, 6865, 6866, 6867, 6868, 6869,6872, 6873, 6875, 6876, 6877, 6878, 6879, 6885, 6888, 6889, 6890, 6891,6892, 6893, 6894, 6895, 6896, 6897, 6898, 6899, 6900, 6901, 6902, 6903,6904, 6905, 6906, 6907, 6908, 6909, 6910, 6911, 6912, 6913, 6914, 6915,6916, 6917, 6918, 6919, 6920, 6921, 6922, 6923, 6924, 6925, 6926, 6927,6928, 6929, 6930, 6931, 6932, 6933, 6934, 6935, 6936, 6953, 6954, 6955,6957, 6958, 6959, 6960, 6962, 6963, 6964, 6965, 6966, 6967, 6968, 6969,6970, 6971, 6972, 6973, 6983, 6984, 6985, 6988, 6989, 6990, 6991, 6992,6993, 6994, 6995, 7007, 7008, 7009, 7010, 7011, 7012, 7015, 7016, 7017,7018, 7019, 7020, 7021, 7034, 7035, 7084, 7086, 7087, 7088, 7089, 7090,7091, 7092, 7128, 7137, 7138, 7139, 7140, 7141, 7142, 7143, 7144, 7145,7146, 7147, 7148, 7149, 7150, 7151, 7152, 7153, 7154, 7155, 7195, 7196,7197, 7198, 7200, 7201, 7202, 7203, 7204, 7205, 7206, 7207, 7208, 7209,7210, 7211, 7212, 7213, 7214, 7215, 7216, 7217, 7218, 7219, 7220, 7221,7222, 7223, 7224, 7225, 7226, 7227, 7228, 7229, 7230, 7231, 7232, 7233,7234, 7235, 7236, 7237, 7238, 7239, 7240, 7241, 7243, 7244, 7245, 7246,7247, 7248, 7249, 7250, 7251, 7252, 7253, 7254, 7255, 7256, 7257, 7258,7259, 7260, 7261, 7262, 7263, 7264, 7265, 7266, 7267, 7268, 7269, 7270,7271, 7291, 7292, 7293, 7294, 7295, 7296, 7297, 7298, 7347, 7348, 7349,7358, 7359, 7360, 7361, 7362, 7363, 7364, 7365, 7372, 7373, 7374, 7375,7376, 7377, 7378, 7383, 7415, 7416, 7417, 7438, 7471, 7472, 7473, 7474,7475, 7476, 7477, 7490, 7496, 7497, 7498, 7499, 7500, 7501, 7502, 7503,7504, 7505, 7506, 7507, 7508, 7509, 7512, 7513, 7514, 7515, 7532, 7533,7534, 7535, 7537, 7538, 7539, 7540, 7541, 7542, 7543, 7544, 7545, 7546,7547, 7548, 7549, 7550, 7551, 7552, 7553, 7554, 7555, 7556, 7557, 7558,7559, 7560, 7585, 7586, 7587, 7588, 7589, 7590, 7591, 7592, 7593, 7595,7596, 7597, 7599, 7600, 7601, 7617, 7618, 7619, 7620, 7621, 7622, 7623,7638, 7668, 7669, 7670, 7672, 7673, 7674, 7675, 7676, 7685, 7687, 7694,7695, 7696, 7717, 7718, 7719, 7720, 7721, 7722, 7723, 7724, 7725, 7726,7727, 7728, 7729, 7730, 7731, 7732, 7733, 7734, 7735, 7736, 7737, 7738,7739, 7740, 7741, 7742, 7743, 7744, 7746, 7747, 7748, 7749, 7750, 7751,7752, 7757, 7758, 7759, 7760, 7761, 7762, 7763, 7764, 7765, 7766, 7767,7768, 7796, 7797, 7805, 7813, 7814, 7815, 7816, 7817, 7818, 7819, 7820,7821, 7822, 7848, 7849, 7850, 7851, 7852, 7853, 7854, 7859, 7860, 7861,7862, 7863, 7864, 7865, 7866, 7867, 7869, 7870, 7874, 7875, 7917, 7918,7919, 7925, 7926, 7927, 7928, 7929, 7941, 7942, 7943, 7944, 7945, 7946,7947, 7948, 7949, 7950, 7951, 7952, 7953, 7955, 7956, 7957, 7958, 7959,7960, 7961, 7962, 7963, 7964, 7966, 7967, 7968, 7969, 7970, 7971, 7972,7974, 7975, 7976, 7977, 7978, 7979, 7980, 7981, 7982, 7983, 7984, 7985,7986, 7987, 7988, 7989, 7990, 7991, 7992, 7993, 7994, 7995, 7996, 7997,7998, 7999, 8000, 8011, 8012, 8013, 8014, 8015, 8016, 8017, 8018, 8019,8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027, 8028, 8029, 8030, 8048,8049, 8050, 8051, 8052, 8053, 8054, 8055, 8056, 8057, 8087, 8088, 8089,8090, 8091, 8092, 8095, 8096, 8097, 8098, 8099, 8100, 8101, 8102, 8103,8104, 8105, 8106, 8107, 8110, 8111, 8112, 8113, 8114, 8115, 8116, 8117,8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125, 8126, 8127, 8130, 8131,8132, 8133, 8134, 8135, 8136, 8144, 8145, 8166, 8169, 8170, 8171, 8172,8173, 8174, 8175, 8176, 8177, 8178, 8179, 8180, 8181, 8182, 8188, 8191,8192, 8193, 8194, 8195, 8196, 8197, 8198, 8209, 8210, 8211, 8212, 8215,8227, 8230, 8231, 8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240,8241, 8242, 8243, 8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251, 8260,8261, 8262, 8263, 8299, 8302, 8303, 8304, 8305, 8306, 8353, 8354, 8355,8356, 8357, 8358, 8359, 8360, 8361, 8375, 8376, 8377, 8378, 8379, 8380,8381, 8382, 8410, 8411, 8412, 8440, 8459, 8460, 8461, 8462, 8463, 8474,8475, 8476, 8477, 8478, 8508, 8509, 8510, 8511, 8512, 8514, 8515, 8546,8547, 8548, 8549, 8550, 8551, 8552, 8553, 8554, 8555, 8570, 8571, 8572,8573, 8574, 8575, 8576, 8577, 8578, 8579, 8580, 8581, 8586, 8587, 8593,8594, 8595, 8596, 8597, 8601, 8602, 8603, 8604, 8605, 8615, 8617, 8618,8655, 8686, 8709, 8712, 8713, 8714, 8715, 8716, 8717, 8718, 8719, 8720,8721, 8722, 8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730, 8731, 8732,8733, 8734, 8770, 8771, 8788, 8789, 8790, 8791, 8792, 8793, 8794, 8795,8796, 8797, 8798, 8799, 8800, 8801, 8802, 8803, 8804, 8805, 8806, 8810,8811, 8817, 8818, 8819, 8820, 8821, 8822, 8823, 8829, 8830, 8837, 8838,8839, 8840, 8841, 8848, 8849, 8850, 8851, 8854, 8855, 8856, 8857, 8858,8859, 8860, 8861, 8862, 8863, 8864, 8865, 8867, 8868, 8872, 8873, 8874,8875, 8876, 8878, 8879, 8881, 8882, 8883, 8884, 8885, 8886, 8887, 8888,8889, 8890, 8891, 8892, 8893, 8894, 8895, 8896, 8897, 8898, 8899, 8900,8901, 8902, 8903, 8904, 8906, 8913, 8914, 8915, 8916, 8917, 8918, 8919,8920, 8921, 8925, 8926, 8927, 8928, 8933, 8934, 8935, 8936, 8937, 8938,8939, 8940, 8941, 8942, 8943, 8944, 8945, 8946, 8947, 8948, 8949, 8950,8951, 8954, 8955, 8956, 8992, 8993, 8994, 8995, 8996, 8997, 8998, 8999,9000, 9001, 9002, 9003, 9010, 9011, 9012, 9013, 9014, 9018, 9019, 9020,9021, 9022, 9023, 9024, 9025, 9026, 9027, 9028, 9029, 9030, 9031, 9041,9042, 9043, 9044, 9045, 9046, 9047, 9048, 9049, 9050, 9051, 9052, 9053,9054, 9055, 9056, 9057, 9058, 9059, 9065, 9066, 9087, 9088, 9091, 9092,9093, 9094, 9095, 9096, 9097, 9098, 9103, 9104, 9105, 9106, 9107, 9108,9109, 9110, 9123, 9124, 9125, 9126, 9127, 9128, 9129, 9130, 9131, 9133,9134, 9135, 9136, 9137, 9145, 9146, 9147, 9148, 9149, 9150, 9151, 9160,9161, 9162, 9163, 9201, 9203.

Esophageal Cancer

Esophageal cancer is cancer that forms in the tissues lining theesophagus. There are two common types of esophageal cancer which arenamed for the type of cells that become malignant. Squamous cellcarcinoma is cancer that forms in the thin, flat cells lining theesophagus (also called epidermoid carcinoma). Cancer that begins in theglandular (secretory) cells which produce and release fluids such asmucus is called adenocarcinoma. Common symptoms associated withesophageal cancer include, but are not limited to, painful or difficultswallowing, weight loss, pain behind the breastbone, hoarseness andcough, and indigestion and heartburn.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withesophageal cancer by administering to said subject an isolatedpolynucleotide encoding an oncology-related polypeptide of interest. Inone embodiment, the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA of the present inventionmay be used to reduce, eliminate, or prevent tumor growth in a subjectwho has been diagnosed or may be diagnosed with esophageal cancer byadministering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In one embodiment, theoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA of the present invention may be used to reduceand/or ameliorate at least one symptom of cancer in a subject who hasbeen diagnosed or may be diagnosed with esophageal cancer byadministering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In a further embodiment, theoncology-related polypeptide of interest may include, but not limitedto, SEQ ID NOs: 4711, 4712, 4713, 4714, 4715, 4716, 4717, 4718, 4719,4720, 4721, 4722, 4723, 4724, 4725, 4726, 4727, 4728, 4742, 4743, 4744,4745, 4746, 4747, 4748, 4749, 4750, 4751, 4781, 4782, 4783, 4784, 4785,4786, 4787, 4788, 4789, 4790, 4825, 4826, 4827, 4828, 4829, 4830, 4832,4854, 4855, 4856, 4857, 4858, 4859, 4860, 4861, 4863, 4864, 4865, 4866,4867, 4868, 4869, 4870, 4871, 4872, 4873, 4885, 4886, 4887, 4888, 4889,4890, 4891, 4892, 4893, 4894, 4911, 4912, 4913, 4914, 4915, 4916, 4917,4918, 4919, 4920, 4960, 4961, 4962, 4963, 4964, 4965, 4966, 4967, 4968,4969, 4970, 4971, 4972, 4973, 4974, 4975, 4976, 4977, 4978, 4979, 4980,4981, 4982, 4983, 4984, 4985, 4986, 4987, 4988, 4989, 4990, 4991, 4992,4993, 4994, 4995, 4996, 4997, 4998, 4999, 5000, 5001, 5002, 5008, 5009,5010, 5011, 5012, 5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021,5022, 5023, 5024, 5025, 5026, 5027, 5028, 5030, 5031, 5032, 5033, 5034,5035, 5036, 5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046,5047, 5048, 5049, 5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058,5059, 5060, 5061, 5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070,5071, 5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079, 5103, 5104, 5105,5106, 5107, 5108, 5109, 5110, 5118, 5119, 5120, 5121, 5122, 5123, 5124,5125, 5126, 5127, 5128, 5129, 5130, 5131, 5132, 5133, 5134, 5135, 5136,5137, 5138, 5139, 5140, 5141, 5145, 5146, 5147, 5148, 5149, 5150, 5151,5152, 5153, 5154, 5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163,5164, 5165, 5166, 5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175,5176, 5177, 5178, 5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190,5191, 5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202,5203, 5204, 5205, 5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5230,5231, 5252, 5253, 5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262,5263, 5264, 5265, 5266, 5267, 5268, 5269, 5270, 5271, 5291, 5292, 5293,5294, 5295, 5296, 5297, 5298, 5299, 5300, 5301, 5302, 5303, 5304, 5305,5306, 5307, 5308, 5309, 5310, 5311, 5312, 5313, 5314, 5315, 5316, 5317,5318, 5319, 5320, 5321, 5322, 5323, 5324, 5325, 5326, 5327, 5328, 5329,5330, 5331, 5332, 5333, 5334, 5335, 5336, 5337, 5338, 5339, 5340, 5341,5342, 5343, 5344, 5345, 5346, 5347, 5348, 5349, 5350, 5351, 5352, 5353,5354, 5355, 5356, 5357, 5358, 5359, 5360, 5361, 5362, 5363, 5364, 5365,5366, 5367, 5368, 5369, 5370, 5371, 5372, 5373, 5374, 5375, 5376, 5377,5378, 5379, 5380, 5381, 5382, 5383, 5384, 5385, 5386, 5387, 5388, 5389,5390, 5391, 5392, 5393, 5419, 5420, 5421, 5426, 5427, 5428, 5429, 5438,5447, 5448, 5449, 5450, 5451, 5452, 5453, 5454, 5455, 5456, 5457, 5458,5459, 5460, 5461, 5462, 5463, 5464, 5465, 5469, 5470, 5477, 5478, 5479,5480, 5481, 5482, 5490, 5491, 5492, 5493, 5494, 5495, 5496, 5497, 5498,5499, 5504, 5505, 5506, 5507, 5508, 5509, 5510, 5511, 5512, 5513, 5514,5515, 5516, 5517, 5518, 5519, 5520, 5521, 5522, 5523, 5524, 5525, 5526,5527, 5528, 5529, 5530, 5531, 5532, 5533, 5534, 5535, 5536, 5548, 5549,5551, 5552, 5553, 5554, 5555, 5556, 5557, 5558, 5559, 5560, 5561, 5562,5563, 5564, 5565, 5566, 5567, 5568, 5569, 5570, 5571, 5577, 5578, 5579,5580, 5590, 5591, 5592, 5593, 5594, 5595, 5596, 5605, 5606, 5607, 5608,5609, 5610, 5611, 5616, 5617, 5618, 5619, 5620, 5621, 5622, 5623, 5631,5632, 5633, 5638, 5639, 5640, 5671, 5672, 5673, 5674, 5675, 5676, 5677,5678, 5679, 5680, 5681, 5688, 5689, 5690, 5691, 5692, 5693, 5694, 5695,5696, 5697, 5698, 5699, 5700, 5701, 5702, 5703, 5708, 5709, 5710, 5711,5712, 5713, 5714, 5715, 5716, 5717, 5727, 5740, 5741, 5743, 5744, 5745,5746, 5747, 5748, 5749, 5750, 5751, 5755, 5757, 5758, 5759, 5760, 5761,5762, 5763, 5764, 5765, 5766, 5767, 5768, 5769, 5770, 5772, 5773, 5775,5776, 5777, 5778, 5779, 5780, 5783, 5784, 5785, 5786, 5787, 5788, 5789,5790, 5791, 5792, 5793, 5794, 5795, 5796, 5797, 5798, 5799, 5800, 5801,5802, 5803, 5811, 5812, 5813, 5814, 5821, 5822, 5823, 5824, 5825, 5826,5827, 5828, 5829, 5830, 5831, 5833, 5834, 5836, 5837, 5838, 5839, 5848,5849, 5850, 5851, 5852, 5853, 5854, 5855, 5864, 5865, 5866, 5867, 5868,5875, 5876, 5877, 5878, 5880, 5881, 5882, 5883, 5884, 5885, 5886, 5887,5888, 5889, 5890, 5891, 5892, 5893, 5894, 5895, 5896, 5897, 5898, 5899,5907, 5908, 5909, 5911, 5912, 5913, 5914, 5915, 5916, 5917, 5918, 5919,5920, 5921, 5922, 5929, 5950, 5951, 5952, 5953, 5954, 5955, 5956, 5963,5967, 5969, 5970, 5982, 5983, 5984, 5985, 5986, 5987, 5988, 5997, 5998,5999, 6000, 6001, 6002, 6068, 6069, 6070, 6071, 6072, 6074, 6075, 6076,6077, 6078, 6080, 6081, 6082, 6083, 6084, 6085, 6086, 6087, 6088, 6089,6090, 6091, 6092, 6093, 6094, 6095, 6096, 6097, 6098, 6100, 6101, 6102,6103, 6105, 6106, 6107, 6108, 6109, 6110, 6111, 6112, 6113, 6114, 6115,6116, 6117, 6118, 6119, 6120, 6121, 6122, 6123, 6124, 6128, 6130, 6142,6143, 6144, 6145, 6146, 6147, 6148, 6149, 6151, 6152, 6153, 6154, 6155,6156, 6157, 6169, 6170, 6171, 6172, 6173, 6174, 6175, 6176, 6177, 6178,6179, 6180, 6181, 6182, 6185, 6186, 6187, 6191, 6208, 6209, 6231, 6232,6233, 6234, 6235, 6236, 6237, 6238, 6239, 6251, 6256, 6257, 6258, 6259,6260, 6261, 6262, 6278, 6279, 6280, 6281, 6282, 6286, 6287, 6288, 6289,6290, 6298, 6299, 6300, 6301, 6302, 6303, 6304, 6305, 6306, 6307, 6308,6309, 6310, 6311, 6312, 6313, 6314, 6315, 6356, 6357, 6358, 6378, 6379,6387, 6388, 6389, 6390, 6391, 6392, 6393, 6394, 6395, 6396, 6397, 6398,6399, 6400, 6401, 6402, 6403, 6405, 6406, 6407, 6451, 6452, 6453, 6497,6498, 6499, 6500, 6501, 6502, 6503, 6509, 6510, 6511, 6512, 6514, 6519,6520, 6521, 6522, 6523, 6549, 6550, 6551, 6552, 6553, 6554, 6555, 6556,6557, 6558, 6588, 6589, 6599, 6600, 6601, 6602, 6603, 6604, 6605, 6606,6611, 6612, 6613, 6614, 6615, 6616, 6617, 6618, 6619, 6620, 6621, 6622,6623, 6624, 6625, 6626, 6627, 6628, 6629, 6630, 6631, 6632, 6633, 6634,6638, 6639, 6640, 6641, 6642, 6643, 6644, 6684, 6685, 6686, 6697, 6698,6699, 6700, 6701, 6719, 6721, 6723, 6724, 6754, 6755, 6756, 6757, 6758,6759, 6760, 6761, 6762, 6763, 6764, 6796, 6797, 6798, 6799, 6800, 6805,6806, 6807, 6808, 6809, 6814, 6815, 6816, 6817, 6818, 6824, 6825, 6826,6831, 6832, 6833, 6834, 6853, 6854, 6855, 6856, 6857, 6860, 6861, 6862,6863, 6864, 6865, 6866, 6867, 6868, 6869, 6872, 6873, 6875, 6876, 6877,6878, 6879, 6885, 6888, 6889, 6890, 6891, 6892, 6893, 6894, 6895, 6896,6897, 6898, 6899, 6900, 6901, 6902, 6903, 6904, 6905, 6906, 6907, 6908,6909, 6910, 6911, 6912, 6913, 6914, 6915, 6916, 6917, 6918, 6919, 6920,6921, 6922, 6923, 6924, 6925, 6926, 6927, 6928, 6929, 6930, 6931, 6932,6933, 6934, 6935, 6936, 6953, 6954, 6955, 6957, 6958, 6959, 6960, 6962,6963, 6964, 6965, 6966, 6967, 6968, 6969, 6970, 6971, 6972, 6973, 6983,6984, 6985, 6988, 6989, 6990, 6991, 6992, 6993, 6994, 6995, 7007, 7008,7009, 7010, 7011, 7012, 7015, 7016, 7017, 7018, 7019, 7020, 7021, 7034,7035, 7084, 7086, 7087, 7088, 7089, 7090, 7091, 7092, 7128, 7137, 7138,7139, 7140, 7141, 7142, 7143, 7144, 7145, 7146, 7147, 7148, 7149, 7150,7151, 7152, 7153, 7154, 7155, 7195, 7196, 7197, 7198, 7200, 7201, 7202,7203, 7204, 7205, 7206, 7207, 7208, 7209, 7210, 7211, 7212, 7213, 7214,7215, 7216, 7217, 7218, 7219, 7220, 7221, 7222, 7223, 7224, 7225, 7226,7227, 7228, 7229, 7230, 7231, 7232, 7233, 7234, 7235, 7236, 7237, 7238,7239, 7240, 7241, 7243, 7244, 7245, 7246, 7247, 7248, 7249, 7250, 7251,7252, 7253, 7254, 7255, 7256, 7257, 7258, 7259, 7260, 7261, 7262, 7263,7264, 7265, 7266, 7267, 7268, 7269, 7270, 7271, 7291, 7292, 7293, 7294,7295, 7296, 7297, 7298, 7347, 7348, 7349, 7358, 7359, 7360, 7361, 7362,7363, 7364, 7365, 7372, 7373, 7374, 7375, 7376, 7377, 7378, 7383, 7415,7416, 7417, 7438, 7471, 7472, 7473, 7474, 7475, 7476, 7477, 7490, 7496,7497, 7498, 7499, 7500, 7501, 7502, 7503, 7504, 7505, 7506, 7507, 7508,7509, 7510, 7511, 7512, 7513, 7514, 7515, 7532, 7533, 7534, 7535, 7537,7538, 7539, 7540, 7541, 7542, 7543, 7544, 7545, 7546, 7547, 7548, 7549,7550, 7551, 7552, 7553, 7554, 7555, 7556, 7557, 7558, 7559, 7560, 7585,7586, 7587, 7588, 7589, 7590, 7591, 7592, 7593, 7595, 7596, 7597, 7599,7600, 7601, 7617, 7618, 7619, 7620, 7621, 7622, 7623, 7638, 7668, 7669,7670, 7672, 7673, 7674, 7675, 7676, 7685, 7687, 7694, 7695, 7696, 7717,7718, 7719, 7720, 7721, 7722, 7723, 7724, 7725, 7726, 7727, 7728, 7729,7730, 7731, 7732, 7733, 7734, 7735, 7736, 7737, 7738, 7739, 7740, 7741,7742, 7743, 7744, 7746, 7747, 7748, 7749, 7750, 7751, 7752, 7757, 7758,7759, 7760, 7761, 7762, 7763, 7764, 7765, 7766, 7767, 7768, 7796, 7797,7805, 7813, 7814, 7815, 7816, 7817, 7818, 7819, 7820, 7821, 7822, 7848,7849, 7850, 7851, 7852, 7853, 7854, 7859, 7860, 7861, 7862, 7863, 7864,7865, 7866, 7867, 7869, 7870, 7874, 7875, 7917, 7918, 7919, 7925, 7926,7927, 7928, 7929, 7941, 7942, 7943, 7944, 7945, 7946, 7947, 7948, 7949,7950, 7951, 7952, 7953, 7955, 7956, 7957, 7958, 7959, 7960, 7961, 7962,7963, 7964, 7965, 7966, 7967, 7968, 7969, 7970, 7971, 7972, 7974, 7975,7976, 7977, 7978, 7979, 7980, 7981, 7982, 7983, 7984, 7985, 7986, 7987,7988, 7989, 7990, 7991, 7992, 7993, 7994, 7995, 7996, 7997, 7998, 7999,8000, 8011, 8012, 8013, 8014, 8015, 8016, 8017, 8018, 8019, 8020, 8021,8022, 8023, 8024, 8025, 8026, 8027, 8028, 8029, 8030, 8048, 8049, 8050,8051, 8052, 8053, 8054, 8055, 8056, 8057, 8087, 8088, 8089, 8090, 8091,8092, 8095, 8096, 8097, 8098, 8099, 8100, 8101, 8102, 8103, 8104, 8105,8106, 8107, 8110, 8111, 8112, 8113, 8114, 8115, 8116, 8117, 8118, 8119,8120, 8121, 8122, 8123, 8124, 8125, 8126, 8127, 8130, 8131, 8132, 8133,8134, 8135, 8136, 8144, 8145, 8166, 8169, 8170, 8171, 8172, 8173, 8174,8175, 8176, 8177, 8178, 8179, 8180, 8181, 8182, 8188, 8191, 8192, 8193,8194, 8195, 8196, 8197, 8198, 8199, 8200, 8201, 8209, 8210, 8211, 8212,8215, 8227, 8230, 8231, 8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239,8240, 8241, 8242, 8243, 8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251,8260, 8261, 8262, 8263, 8299, 8302, 8303, 8304, 8305, 8306, 8353, 8354,8355, 8356, 8357, 8358, 8359, 8360, 8361, 8375, 8376, 8377, 8378, 8379,8380, 8381, 8382, 8410, 8411, 8412, 8440, 8459, 8460, 8461, 8462, 8463,8474, 8475, 8476, 8477, 8478, 8508, 8509, 8510, 8511, 8512, 8514, 8515,8546, 8547, 8548, 8549, 8550, 8551, 8552, 8553, 8554, 8555, 8570, 8571,8572, 8573, 8574, 8575, 8576, 8577, 8578, 8579, 8580, 8581, 8586, 8587,8593, 8594, 8595, 8596, 8597, 8601, 8602, 8603, 8604, 8605, 8615, 8617,8618, 8655, 8686, 8709, 8712, 8713, 8714, 8715, 8716, 8717, 8718, 8719,8720, 8721, 8722, 8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730, 8731,8732, 8733, 8734, 8770, 8771, 8788, 8789, 8790, 8791, 8792, 8793, 8794,8795, 8796, 8797, 8798, 8799, 8800, 8801, 8802, 8803, 8804, 8805, 8806,8810, 8811, 8817, 8818, 8819, 8820, 8821, 8822, 8823, 8829, 8830, 8837,8838, 8839, 8840, 8841, 8848, 8849, 8850, 8851, 8854, 8855, 8856, 8857,8858, 8859, 8860, 8861, 8862, 8863, 8864, 8865, 8867, 8868, 8872, 8873,8874, 8875, 8876, 8878, 8879, 8880, 8881, 8882, 8883, 8884, 8885, 8886,8887, 8888, 8889, 8890, 8891, 8892, 8893, 8894, 8895, 8896, 8897, 8898,8899, 8900, 8901, 8902, 8903, 8904, 8906, 8913, 8914, 8915, 8916, 8917,8918, 8919, 8920, 8921, 8925, 8926, 8927, 8928, 8933, 8934, 8935, 8936,8937, 8938, 8939, 8940, 8941, 8942, 8943, 8944, 8945, 8946, 8947, 8948,8949, 8950, 8951, 8954, 8955, 8956, 8992, 8993, 8994, 8995, 8996, 8997,8998, 8999, 9000, 9001, 9002, 9003, 9010, 9011, 9012, 9013, 9014, 9018,9019, 9020, 9021, 9022, 9023, 9024, 9025, 9026, 9027, 9028, 9029, 9030,9031, 9041, 9042, 9043, 9044, 9045, 9046, 9047, 9048, 9049, 9050, 9051,9052, 9053, 9054, 9055, 9056, 9057, 9058, 9059, 9065, 9066, 9087, 9088,9091, 9092, 9093, 9094, 9095, 9096, 9097, 9098, 9103, 9104, 9105, 9106,9107, 9108, 9109, 9110, 9123, 9124, 9125, 9126, 9127, 9128, 9129, 9130,9131, 9133, 9134, 9135, 9136, 9137, 9145, 9146, 9147, 9148, 9149, 9150,9151, 9160, 9161, 9162, 9163, 9201, 9203.

Familial Cancer Syndrome

Familial cancer syndrome describes the genetic predisposition of asubject to develop cancer. 5-10% of all cancers are hereditary and arepassed on through specific in specific genes passed from one bloodrelative to another. Subjects that inherit one of these gene changes mayhave a higher likelihood of developing cancer within their lifetime.Familial cancer syndrome includes disorder such as, but not limited to,Ataxia Telangiectasia, Basal Cell Nevus Syndrome, Nevoid Basal CellCarcinoma Syndrome, Gorlin Syndrome, Beck-with Wiedemann Syndrome,Birt-Hogg-Dube Syndrome, Bloom Syndrome, hereditary breast and/orovarian cancer, Carney Complex, Types I and II, Familial Chordoma, ColonCancer, Hereditary Nonpolyposis-Lynch Syndrome, Costello Syndrome,Facio-Cutaneous-Skeletal Syndrome, Cowden Syndrome, DyskeratosisCongenita, Tylosis with Esophaeal Cancer, Keratosis Palmaris etPlantaris with Esophageal Cancer, Howel-Evans Syndrome, HerediataryMultiple Exostosis, Fanconi Anemia, Hereditary Diffuse Gastric Cancer,Gastrointestinal Stromal Tumor, Multiple Gastrointestinal Stromal Tumor,Familial Hyperparathyroidism, Acute Myeloid Leukemia, Familial Leukemia,Chronic Lymphocytic Leukemia, Li-Fraumeni Syndrome, Hodgkin Lymphoma,Non-Hodgkin Lymphoma, Hereditary Multiple Melanoma, Mosaic VarigatedAneuploidy, Multiple Endocrine Neoplasia Type I, Type 2A and 2B,Familial Medullary Thyroid Cancer, Familial Multiple Myeloma, HereditaryNeuroblastoma, Neurofibromatosis Type 1 and 2, Nijmegen BreakageSyndrome, Hereditary Pancreatic Cancer, Hereditary Paraganglioma,Peutz-Jeghers Syndrome, Familial Adenomatous Polyposis, FamilialJuvenile Polyposis, MYH-Associated Polyposis, Hereditary ProstateCancer, Hereditary Renal Cell Carcinoma with Multiple Cutaneous andUterine Leiomyomas, Hereditary Renal Cell Carcinoma, HereditaryPapillary Renal Cell Carcinoma, Rhabdoid Predisposition Syndrome,Rothmund-Thomson Syndrome, Simpson-Golabi-Behmel Syndrome, FamilialTesticular Germ Cell Tumor, Familial Non-medullary Thyroid Carcinoma,Tuberous Sclerosis Complex, von Hippel-Lindau Syndrome, FamilialWaldenstrom Macroglobulinemia, Werner Syndrome, Familial Wilms Tumor andXeroderma Pigmentosum.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withFamilial cancer syndrome by administering to said subject an isolatedpolynucleotide encoding an oncology-related polypeptide of interest. Inone embodiment, the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA of the present inventionmay be used to reduce, eliminate, or prevent tumor growth in a subjectwho has been diagnosed or may be diagnosed with Familial cancer syndromeby administering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In one embodiment, theoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA of the present invention may be used to reduceand/or ameliorate at least one symptom of cancer in a subject who hasbeen diagnosed or may be diagnosed with Familial cancer syndrome byadministering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In a further embodiment, theoncology-related polypeptide of interest may include, but not limitedto, SEQ ID NOs: 4704, 4705, 4706, 4707, 4708, 4709, 4710, 4711, 4712,4713, 4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722, 4723, 4724,4725, 4726, 4727, 4728, 4729, 4730, 4731, 4732, 4733, 4734, 4735, 4736,4737, 4738, 4739, 4740, 4741, 4742, 4743, 4744, 4745, 4746, 4747, 4748,4749, 4750, 4751, 4752, 4753, 4754, 4755, 4756, 4757, 4758, 4759, 4760,4761, 4762, 4763, 4764, 4765, 4766, 4767, 4768, 4769, 4770, 4771, 4772,4773, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784,4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796,4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4808,4809, 4810, 4811, 4812, 4813, 4814, 4815, 4816, 4817, 4818, 4819, 4820,4821, 4822, 4823, 4824, 4825, 4826, 4827, 4828, 4829, 4830, 4831, 4832,4833, 4834, 4835, 4836, 4837, 4838, 4839, 4840, 4841, 4842, 4843, 4844,4845, 4846, 4847, 4848, 4849, 4850, 4851, 4852, 4853, 4854, 4855, 4856,4857, 4858, 4859, 4860, 4861, 4862, 4863, 4864, 4865, 4866, 4867, 4868,4869, 4870, 4871, 4872, 4873, 4874, 4875, 4876, 4877, 4878, 4879, 4880,4881, 4882, 4883, 4884, 4885, 4886, 4887, 4888, 4889, 4890, 4891, 4892,4893, 4894, 4895, 4896, 4897, 4898, 4899, 4900, 4901, 4902, 4903, 4904,4905, 4906, 4907, 4908, 4909, 4910, 4911, 4912, 4913, 4914, 4915, 4916,4917, 4918, 4919, 4920, 4921, 4922, 4923, 4924, 4925, 4926, 4927, 4928,4929, 4930, 4931, 4932, 4933, 4934, 4935, 4936, 4937, 4938, 4939, 4940,4941, 4942, 4943, 4944, 4945, 4946, 4947, 4948, 4949, 4950, 4951, 4952,4953, 4954, 4955, 4956, 4957, 4958, 4959, 4960, 4961, 4962, 4963, 4964,4965, 4966, 4967, 4968, 4969, 4970, 4971, 4972, 4973, 4974, 4975, 4976,4977, 4978, 4979, 4980, 4981, 4982, 4983, 4984, 4985, 4986, 4987, 4988,4989, 4990, 4991, 4992, 4993, 4994, 4995, 4996, 4997, 4998, 4999, 5000,5001, 5002, 5003, 5004, 5005, 5006, 5007, 5008, 5009, 5010, 5011, 5012,5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021, 5022, 5023, 5024,5025, 5026, 5027, 5028, 5029, 5030, 5031, 5032, 5033, 5034, 5035, 5036,5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046, 5047, 5048,5049, 5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058, 5059, 5060,5061, 5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071, 5072,5073, 5074, 5075, 5076, 5077, 5078, 5079, 5080, 5081, 5082, 5083, 5084,5085, 5086, 5087, 5088, 5089, 5090, 5091, 5092, 5093, 5094, 5095, 5096,5097, 5098, 5099, 5100, 5101, 5102, 5103, 5104, 5105, 5106, 5107, 5108,5109, 5110, 5111, 5112, 5113, 5114, 5115, 5116, 5117, 5118, 5119, 5120,5121, 5122, 5123, 5124, 5125, 5126, 5127, 5128, 5129, 5130, 5131, 5132,5133, 5134, 5135, 5136, 5137, 5138, 5139, 5140, 5141, 5142, 5143, 5144,5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152, 5153, 5154, 5155, 5156,5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164, 5165, 5166, 5167, 5168,5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176, 5177, 5178, 5179, 5180,5181, 5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191, 5192,5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203, 5204,5205, 5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5214, 5215, 5216,5217, 5218, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, 5227, 5228,5229, 5230, 5231, 5232, 5233, 5234, 5235, 5236, 5237, 5238, 5239, 5240,5241, 5242, 5243, 5244, 5245, 5246, 5247, 5248, 5249, 5250, 5251, 5252,5253, 5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264,5265, 5266, 5267, 5268, 5269, 5270, 5271, 5272, 5273, 5274, 5275, 5276,5277, 5278, 5279, 5280, 5281, 5282, 5283, 5284, 5285, 5286, 5287, 5288,5289, 5290, 5291, 5292, 5293, 5294, 5295, 5296, 5297, 5298, 5299, 5300,5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311, 5312,5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320, 5321, 5322, 5323, 5324,5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332, 5333, 5334, 5335, 5336,5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344, 5345, 5346, 5347, 5348,5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358, 5359, 5360,5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370, 5371, 5372,5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380, 5381, 5382, 5383, 5384,5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392, 5393, 5394, 5395, 5396,5397, 5398, 5399, 5400, 5401, 5402, 5403, 5404, 5405, 5406, 5407, 5408,5409, 5410, 5411, 5412, 5413, 5414, 5415, 5416, 5417, 5418, 5419, 5420,5421, 5422, 5423, 5424, 5425, 5426, 5427, 5428, 5429, 5430, 5431, 5432,5433, 5434, 5435, 5436, 5437, 5438, 5439, 5440, 5441, 5442, 5443, 5444,5445, 5446, 5447, 5448, 5449, 5450, 5451, 5452, 5453, 5454, 5455, 5456,5457, 5458, 5459, 5460, 5461, 5462, 5463, 5464, 5465, 5466, 5467, 5468,5469, 5470, 5471, 5472, 5473, 5474, 5475, 5476, 5477, 5478, 5479, 5480,5481, 5482, 5483, 5484, 5485, 5486, 5487, 5488, 5489, 5490, 5491, 5492,5493, 5494, 5495, 5496, 5497, 5498, 5499, 5500, 5501, 5502, 5503, 5504,5505, 5506, 5507, 5508, 5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516,5517, 5518, 5519, 5520, 5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528,5529, 5530, 5531, 5532, 5533, 5534, 5535, 5536, 5537, 5538, 5539, 5540,5541, 5542, 5543, 5544, 5545, 5546, 5547, 5548, 5549, 5550, 5551, 5552,5553, 5554, 5555, 5556, 5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564,5565, 5566, 5567, 5568, 5569, 5570, 5571, 5572, 5573, 5574, 5575, 5576,5577, 5578, 5579, 5580, 5581, 5582, 5583, 5584, 5585, 5586, 5587, 5588,5589, 5590, 5591, 5592, 5593, 5594, 5595, 5596, 5597, 5598, 5599, 5600,5601, 5602, 5603, 5604, 5605, 5606, 5607, 5608, 5609, 5610, 5611, 5612,5613, 5614, 5615, 5616, 5617, 5618, 5619, 5620, 5621, 5622, 5623, 5624,5625, 5626, 5627, 5628, 5629, 5630, 5631, 5632, 5633, 5634, 5635, 5636,5637, 5638, 5639, 5640, 5641, 5642, 5643, 5644, 5645, 5646, 5647, 5648,5649, 5650, 5651, 5652, 5653, 5654, 5655, 5656, 5657, 5658, 5659, 5660,5661, 5662, 5663, 5664, 5665, 5666, 5667, 5668, 5669, 5670, 5671, 5672,5673, 5674, 5675, 5676, 5677, 5678, 5679, 5680, 5681, 5682, 5683, 5684,5685, 5686, 5687, 5688, 5689, 5690, 5691, 5692, 5693, 5694, 5695, 5696,5697, 5698, 5699, 5700, 5701, 5702, 5703, 5704, 5705, 5706, 5707, 5708,5709, 5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717, 5718, 5719, 5720,5721, 5722, 5723, 5724, 5725, 5726, 5727, 5728, 5729, 5730, 5731, 5732,5733, 5734, 5735, 5736, 5737, 5738, 5739, 5740, 5741, 5742, 5743, 5744,5745, 5746, 5747, 5748, 5749, 5750, 5751, 5752, 5753, 5754, 5755, 5756,5757, 5758, 5759, 5760, 5761, 5762, 5763, 5764, 5765, 5766, 5767, 5768,5769, 5770, 5771, 5772, 5773, 5774, 5775, 5776, 5777, 5778, 5779, 5780,5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792,5793, 5794, 5795, 5796, 5797, 5798, 5799, 5800, 5801, 5802, 5803, 5804,5805, 5806, 5807, 5808, 5809, 5810, 5811, 5812, 5813, 5814, 5815, 5816,5817, 5818, 5819, 5820, 5821, 5822, 5823, 5824, 5825, 5826, 5827, 5828,5829, 5830, 5831, 5832, 5833, 5834, 5835, 5836, 5837, 5838, 5839, 5840,5841, 5842, 5843, 5844, 5845, 5846, 5847, 5848, 5849, 5850, 5851, 5852,5853, 5854, 5855, 5856, 5857, 5858, 5859, 5860, 5861, 5862, 5863, 5864,5865, 5866, 5867, 5868, 5869, 5870, 5871, 5872, 5873, 5874, 5875, 5876,5877, 5878, 5879, 5880, 5881, 5882, 5883, 5884, 5885, 5886, 5887, 5888,5889, 5890, 5891, 5892, 5893, 5894, 5895, 5896, 5897, 5898, 5899, 5900,5901, 5902, 5903, 5904, 5905, 5906, 5907, 5908, 5909, 5910, 5911, 5912,5913, 5914, 5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922, 5923, 5924,5925, 5926, 5927, 5928, 5929, 5930, 5931, 5932, 5933, 5934, 5935, 5936,5937, 5938, 5939, 5940, 5941, 5942, 5943, 5944, 5945, 5946, 5947, 5948,5949, 5950, 5951, 5952, 5953, 5954, 5955, 5956, 5957, 5958, 5959, 5960,5961, 5962, 5963, 5964, 5965, 5966, 5967, 5968, 5969, 5970, 5971, 5972,5973, 5974, 5975, 5976, 5977, 5978, 5979, 5980, 5981, 5982, 5983, 5984,5985, 5986, 5987, 5988, 5989, 5990, 5991, 5992, 5993, 5994, 5995, 5996,5997, 5998, 5999, 6000, 6001, 6002, 6003, 6004, 6005, 6006, 6007, 6008,6009, 6010, 6011, 6012, 6013, 6014, 6015, 6016, 6017, 6018, 6019, 6020,6021, 6022, 6023, 6024, 6025, 6026, 6027, 6028, 6029, 6030, 6031, 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8720,8721, 8722, 8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730, 8731, 8732,8733, 8734, 8735, 8736, 8737, 8738, 8739, 8740, 8741, 8742, 8743, 8744,8745, 8746, 8747, 8748, 8749, 8750, 8751, 8752, 8753, 8754, 8755, 8756,8757, 8758, 8759, 8760, 8761, 8762, 8763, 8764, 8765, 8766, 8767, 8768,8769, 8770, 8771, 8772, 8773, 8774, 8775, 8776, 8777, 8778, 8779, 8780,8781, 8782, 8783, 8784, 8785, 8786, 8787, 8788, 8789, 8790, 8791, 8792,8793, 8794, 8795, 8796, 8797, 8798, 8799, 8800, 8801, 8802, 8803, 8804,8805, 8806, 8807, 8808, 8809, 8810, 8811, 8812, 8813, 8814, 8815, 8816,8817, 8818, 8819, 8820, 8821, 8822, 8823, 8824, 8825, 8826, 8827, 8828,8829, 8830, 8831, 8832, 8833, 8834, 8835, 8836, 8837, 8838, 8839, 8840,8841, 8842, 8843, 8844, 8845, 8846, 8847, 8848, 8849, 8850, 8851, 8852,8853, 8854, 8855, 8856, 8857, 8858, 8859, 8860, 8861, 8862, 8863, 8864,8865, 8866, 8867, 8868, 8869, 8870, 8871, 8872, 8873, 8874, 8875, 8876,8877, 8878, 8879, 8880, 8881, 8882, 8883, 8884, 8885, 8886, 8887, 8888,8889, 8890, 8891, 8892, 8893, 8894, 8895, 8896, 8897, 8898, 8899, 8900,8901, 8902, 8903, 8904, 8905, 8906, 8907, 8908, 8909, 8910, 8911, 8912,8913, 8914, 8915, 8916, 8917, 8918, 8919, 8920, 8921, 8922, 8923, 8924,8925, 8926, 8927, 8928, 8929, 8930, 8931, 8932, 8933, 8934, 8935, 8936,8937, 8938, 8939, 8940, 8941, 8942, 8943, 8944, 8945, 8946, 8947, 8948,8949, 8950, 8951, 8952, 8953, 8954, 8955, 8956, 8957, 8958, 8959, 8960,8961, 8962, 8963, 8964, 8965, 8966, 8967, 8968, 8969, 8970, 8971, 8972,8973, 8974, 8975, 8976, 8977, 8978, 8979, 8980, 8981, 8982, 8983, 8984,8985, 8986, 8987, 8988, 8989, 8990, 8991, 8992, 8993, 8994, 8995, 8996,8997, 8998, 8999, 9000, 9001, 9002, 9003, 9004, 9005, 9006, 9007, 9008,9009, 9010, 9011, 9012, 9013, 9014, 9015, 9016, 9017, 9018, 9019, 9020,9021, 9022, 9023, 9024, 9025, 9026, 9027, 9028, 9029, 9030, 9031, 9032,9033, 9034, 9035, 9036, 9037, 9038, 9039, 9040, 9041, 9042, 9043, 9044,9045, 9046, 9047, 9048, 9049, 9050, 9051, 9052, 9053, 9054, 9055, 9056,9057, 9058, 9059, 9060, 9061, 9062, 9063, 9064, 9065, 9066, 9067, 9068,9069, 9070, 9071, 9072, 9073, 9074, 9075, 9076, 9077, 9078, 9079, 9080,9081, 9082, 9083, 9084, 9085, 9086, 9087, 9088, 9089, 9090, 9091, 9092,9093, 9094, 9095, 9096, 9097, 9098, 9099, 9100, 9101, 9102, 9103, 9104,9105, 9106, 9107, 9108, 9109, 9110, 9111, 9112, 9113, 9114, 9115, 9116,9117, 9118, 9119, 9120, 9121, 9122, 9123, 9124, 9125, 9126, 9127, 9128,9129, 9130, 9131, 9132, 9133, 9134, 9135, 9136, 9137, 9138, 9139, 9140,9141, 9142, 9143, 9144, 9145, 9146, 9147, 9148, 9149, 9150, 9151, 9152,9153, 9154, 9155, 9156, 9157, 9158, 9159, 9160, 9161, 9162, 9163, 9164,9165, 9166, 9167, 9168, 9169, 9170, 9171, 9172, 9173, 9174, 9175, 9176,9177, 9178, 9179, 9180, 9181, 9182, 9183, 9184, 9185, 9186, 9187, 9188,9189, 9190, 9191, 9192, 9193, 9194, 9195, 9196, 9197, 9198, 9199, 9200,9201, 9202, 9203.

Leukemia

Leukemia is a form of cancer that starts in blood-forming tissue such asthe bone marrow which can cause a large number of blood cells to beproduced and enter the blood stream. Leukemia can also spread to thecentral nervous system and cause brain and spinal cord cancer. Types ofleukemia include, but are not limited to, adult acute lymphoblastic,childhood acute lymphoblastic, aduct acute myeloid, chronic lymphocytic,chronic myelogenous and hairy cell. Non-limiting examples of symptoms ofleukemia include weakness or feeling tired, fever, easy bruising orbleeding, petechiae, shortness of breath, weight loss or loss ofappetite, pain in the bones or stomach, pain or feeling of fullnessbelow the ribs, and painless lumps in the neck, underarm, stomach orgroin.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withleukemia by administering to said subject an isolated polynucleotideencoding an oncology-related polypeptide of interest. In one embodiment,the oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA of the present invention may beused to reduce, eliminate, or prevent tumor growth in a subject who hasbeen diagnosed or may be diagnosed with leukemia by administering tosaid subject an isolated polynucleotide encoding an oncology-relatedpolypeptide of interest. In one embodiment, the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA of the present invention may be used to reduce and/or ameliorateat least one symptom of cancer in a subject who has been diagnosed ormay be diagnosed with leukemia by administering to said subject anisolated polynucleotide encoding an oncology-related polypeptide ofinterest. In a further embodiment, the oncology-related polypeptide ofinterest may include, but not limited to, SEQ ID NOs: 4711, 4712, 4713,4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722, 4723, 4724, 4725,4726, 4727, 4728, 4742, 4743, 4744, 4745, 4746, 4747, 4748, 4749, 4750,4751, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4825,4826, 4827, 4828, 4829, 4830, 4832, 4846, 4854, 4855, 4856, 4857, 4858,4859, 4860, 4861, 4863, 4864, 4868, 4869, 4870, 4871, 4872, 4873, 4874,4875, 4876, 4877, 4878, 4879, 4880, 4881, 4882, 4883, 4884, 4885, 4886,4887, 4888, 4889, 4890, 4891, 4892, 4893, 4894, 4911, 4912, 4913, 4914,4915, 4916, 4917, 4918, 4919, 4920, 4960, 4961, 4962, 4963, 4964, 4965,4966, 4967, 4968, 4969, 4970, 4971, 4972, 4973, 4974, 4975, 4976, 4977,4978, 4979, 4980, 4981, 4982, 4983, 4984, 4985, 4986, 4987, 4988, 4989,4990, 4991, 4992, 4993, 4994, 4995, 4996, 4997, 4998, 4999, 5000, 5001,5002, 5008, 5009, 5010, 5011, 5012, 5013, 5014, 5015, 5016, 5017, 5018,5019, 5020, 5021, 5022, 5023, 5024, 5025, 5026, 5027, 5028, 5030, 5031,5032, 5033, 5034, 5035, 5036, 5037, 5038, 5039, 5040, 5041, 5042, 5043,5044, 5045, 5046, 5047, 5048, 5049, 5050, 5051, 5052, 5053, 5054, 5055,5056, 5057, 5058, 5059, 5060, 5061, 5062, 5063, 5064, 5065, 5066, 5067,5068, 5069, 5070, 5071, 5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079,5093, 5094, 5095, 5096, 5097, 5098, 5099, 5100, 5103, 5104, 5105, 5106,5107, 5108, 5109, 5110, 5111, 5112, 5113, 5114, 5115, 5116, 5117, 5118,5119, 5120, 5121, 5122, 5123, 5124, 5125, 5126, 5127, 5128, 5129, 5130,5131, 5132, 5133, 5134, 5135, 5136, 5137, 5138, 5139, 5140, 5141, 5145,5146, 5147, 5148, 5149, 5150, 5151, 5152, 5153, 5154, 5155, 5156, 5157,5158, 5159, 5160, 5161, 5162, 5163, 5164, 5165, 5166, 5167, 5168, 5169,5170, 5171, 5172, 5173, 5174, 5175, 5176, 5177, 5178, 5179, 5180, 5181,5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191, 5192, 5193,5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203, 5204, 5205,5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5216, 5217, 5218, 5219,5220, 5221, 5222, 5223, 5230, 5231, 5252, 5253, 5254, 5255, 5256, 5257,5258, 5259, 5260, 5261, 5262, 5263, 5264, 5265, 5266, 5267, 5268, 5269,5270, 5271, 5291, 5292, 5293, 5294, 5295, 5296, 5297, 5298, 5299, 5300,5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311, 5312,5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320, 5321, 5322, 5323, 5324,5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332, 5333, 5334, 5335, 5336,5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344, 5345, 5346, 5347, 5348,5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358, 5359, 5360,5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370, 5371, 5372,5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380, 5381, 5382, 5383, 5384,5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392, 5393, 5419, 5420, 5421,5425, 5426, 5427, 5428, 5429, 5438, 5439, 5440, 5441, 5442, 5443, 5447,5448, 5449, 5450, 5451, 5452, 5453, 5454, 5455, 5456, 5457, 5458, 5459,5460, 5461, 5462, 5463, 5464, 5465, 5469, 5470, 5477, 5478, 5479, 5480,5481, 5482, 5490, 5491, 5492, 5493, 5494, 5495, 5496, 5497, 5498, 5499,5500, 5501, 5502, 5503, 5504, 5505, 5506, 5507, 5508, 5509, 5510, 5511,5512, 5513, 5514, 5515, 5516, 5517, 5518, 5519, 5520, 5521, 5522, 5523,5524, 5525, 5526, 5527, 5528, 5529, 5530, 5531, 5532, 5533, 5534, 5535,5536, 5548, 5549, 5551, 5552, 5553, 5554, 5555, 5556, 5557, 5558, 5559,5560, 5561, 5562, 5563, 5564, 5565, 5566, 5567, 5568, 5569, 5570, 5571,5574, 5577, 5578, 5579, 5580, 5590, 5591, 5592, 5593, 5594, 5595, 5596,5605, 5606, 5607, 5608, 5609, 5610, 5611, 5616, 5617, 5618, 5619, 5620,5621, 5622, 5623, 5627, 5628, 5629, 5630, 5631, 5632, 5633, 5638, 5639,5640, 5671, 5672, 5673, 5674, 5675, 5676, 5677, 5678, 5679, 5680, 5681,5688, 5689, 5690, 5691, 5692, 5693, 5694, 5695, 5696, 5697, 5698, 5699,5700, 5701, 5702, 5703, 5708, 5709, 5710, 5711, 5712, 5713, 5714, 5715,5716, 5717, 5727, 5740, 5741, 5743, 5744, 5745, 5746, 5747, 5748, 5749,5750, 5751, 5755, 5757, 5758, 5759, 5760, 5761, 5762, 5763, 5764, 5765,5766, 5767, 5768, 5769, 5770, 5772, 5773, 5775, 5776, 5777, 5778, 5779,5780, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793,5794, 5795, 5796, 5797, 5798, 5799, 5800, 5801, 5802, 5803, 5806, 5807,5808, 5809, 5811, 5812, 5813, 5814, 5821, 5822, 5823, 5824, 5825, 5826,5827, 5828, 5829, 5830, 5831, 5833, 5834, 5836, 5837, 5838, 5839, 5848,5849, 5850, 5851, 5852, 5853, 5854, 5855, 5864, 5865, 5866, 5867, 5868,5875, 5876, 5877, 5878, 5880, 5881, 5882, 5883, 5884, 5885, 5886, 5887,5888, 5889, 5890, 5891, 5892, 5893, 5894, 5895, 5896, 5897, 5898, 5899,5907, 5908, 5909, 5911, 5912, 5913, 5914, 5915, 5916, 5917, 5918, 5919,5920, 5921, 5922, 5923, 5924, 5925, 5926, 5927, 5928, 5929, 5950, 5951,5952, 5953, 5954, 5955, 5956, 5963, 5967, 5969, 5970, 5971, 5972, 5973,5982, 5983, 5984, 5985, 5986, 5987, 5988, 5997, 5998, 5999, 6000, 6001,6002, 6068, 6069, 6070, 6071, 6072, 6074, 6075, 6076, 6077, 6078, 6080,6081, 6082, 6083, 6084, 6085, 6086, 6087, 6088, 6089, 6090, 6091, 6092,6093, 6094, 6095, 6096, 6097, 6098, 6100, 6101, 6102, 6103, 6105, 6106,6107, 6108, 6109, 6110, 6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118,6119, 6120, 6121, 6122, 6123, 6124, 6128, 6130, 6142, 6143, 6144, 6145,6146, 6147, 6148, 6149, 6151, 6152, 6153, 6154, 6155, 6156, 6157, 6169,6170, 6171, 6172, 6173, 6174, 6175, 6176, 6177, 6178, 6179, 6180, 6181,6182, 6185, 6186, 6187, 6191, 6196, 6197, 6198, 6199, 6200, 6201, 6202,6203, 6204, 6205, 6206, 6207, 6208, 6209, 6231, 6232, 6233, 6234, 6235,6236, 6237, 6238, 6239, 6251, 6256, 6257, 6258, 6259, 6260, 6261, 6262,6278, 6279, 6280, 6281, 6282, 6286, 6287, 6288, 6289, 6290, 6298, 6299,6300, 6301, 6302, 6303, 6304, 6305, 6306, 6307, 6308, 6309, 6310, 6311,6312, 6313, 6314, 6315, 6318, 6319, 6320, 6321, 6322, 6323, 6324, 6325,6326, 6356, 6357, 6358, 6378, 6379, 6383, 6384, 6385, 6386, 6387, 6388,6389, 6390, 6391, 6392, 6393, 6394, 6395, 6396, 6397, 6398, 6399, 6400,6401, 6402, 6403, 6405, 6406, 6407, 6422, 6423, 6424, 6425, 6426, 6427,6451, 6452, 6453, 6497, 6498, 6499, 6500, 6501, 6502, 6503, 6509, 6510,6511, 6512, 6514, 6519, 6520, 6521, 6522, 6523, 6533, 6534, 6535, 6536,6537, 6538, 6539, 6540, 6541, 6542, 6543, 6544, 6545, 6546, 6549, 6550,6551, 6552, 6553, 6554, 6555, 6556, 6557, 6558, 6588, 6589, 6599, 6600,6601, 6602, 6603, 6604, 6605, 6606, 6611, 6612, 6613, 6614, 6615, 6616,6617, 6618, 6619, 6620, 6621, 6622, 6623, 6624, 6625, 6626, 6627, 6628,6629, 6630, 6631, 6632, 6633, 6634, 6638, 6639, 6640, 6641, 6642, 6643,6644, 6665, 6666, 6667, 6668, 6669, 6670, 6675, 6676, 6677, 6678, 6679,6680, 6681, 6682, 6683, 6684, 6685, 6686, 6688, 6689, 6697, 6698, 6699,6700, 6701, 6713, 6714, 6715, 6717, 6719, 6721, 6723, 6724, 6754, 6755,6756, 6757, 6758, 6759, 6760, 6761, 6762, 6763, 6764, 6796, 6797, 6798,6799, 6800, 6805, 6806, 6807, 6808, 6809, 6814, 6815, 6816, 6817, 6818,6824, 6825, 6826, 6827, 6828, 6829, 6830, 6831, 6832, 6833, 6834, 6838,6847, 6853, 6854, 6855, 6856, 6857, 6860, 6861, 6862, 6863, 6864, 6865,6866, 6867, 6868, 6869, 6872, 6873, 6875, 6876, 6877, 6878, 6879, 6885,6888, 6889, 6890, 6891, 6892, 6893, 6894, 6895, 6896, 6897, 6898, 6899,6900, 6901, 6902, 6903, 6904, 6905, 6906, 6907, 6908, 6909, 6910, 6911,6912, 6913, 6914, 6915, 6916, 6917, 6918, 6919, 6920, 6921, 6922, 6923,6924, 6925, 6926, 6927, 6928, 6929, 6930, 6931, 6932, 6933, 6934, 6935,6936, 6953, 6954, 6955, 6956, 6957, 6958, 6959, 6960, 6962, 6963, 6964,6965, 6966, 6967, 6968, 6969, 6970, 6971, 6972, 6973, 6983, 6984, 6985,6988, 6989, 6990, 6991, 6992, 6993, 6994, 6995, 7007, 7008, 7009, 7010,7011, 7012, 7015, 7016, 7017, 7018, 7019, 7020, 7021, 7034, 7035, 7084,7086, 7087, 7088, 7089, 7090, 7091, 7092, 7128, 7137, 7138, 7139, 7140,7141, 7142, 7143, 7144, 7145, 7146, 7147, 7148, 7149, 7150, 7151, 7152,7153, 7154, 7155, 7195, 7196, 7197, 7198, 7200, 7201, 7202, 7203, 7204,7205, 7206, 7207, 7208, 7209, 7210, 7211, 7212, 7213, 7214, 7215, 7216,7217, 7218, 7219, 7220, 7221, 7222, 7223, 7224, 7225, 7226, 7227, 7228,7229, 7230, 7231, 7232, 7233, 7234, 7235, 7236, 7237, 7238, 7239, 7240,7241, 7243, 7244, 7245, 7246, 7247, 7248, 7249, 7250, 7251, 7252, 7253,7254, 7255, 7256, 7257, 7258, 7259, 7260, 7261, 7262, 7263, 7264, 7265,7266, 7267, 7268, 7269, 7270, 7271, 7291, 7292, 7293, 7294, 7295, 7296,7297, 7298, 7347, 7348, 7349, 7358, 7359, 7360, 7361, 7362, 7363, 7364,7365, 7372, 7373, 7374, 7375, 7376, 7377, 7378, 7383, 7415, 7416, 7417,7437, 7438, 7458, 7459, 7460, 7461, 7467, 7468, 7469, 7470, 7471, 7472,7473, 7474, 7475, 7476, 7477, 7490, 7496, 7497, 7498, 7499, 7500, 7501,7502, 7503, 7504, 7505, 7506, 7507, 7508, 7509, 7510, 7511, 7512, 7513,7514, 7515, 7532, 7533, 7534, 7535, 7537, 7538, 7539, 7540, 7541, 7542,7543, 7544, 7545, 7546, 7547, 7548, 7549, 7550, 7551, 7552, 7553, 7554,7555, 7556, 7557, 7558, 7559, 7560, 7585, 7586, 7587, 7588, 7589, 7590,7591, 7592, 7593, 7595, 7596, 7597, 7599, 7600, 7601, 7617, 7618, 7619,7620, 7621, 7622, 7623, 7638, 7668, 7669, 7670, 7672, 7673, 7674, 7675,7676, 7685, 7687, 7694, 7695, 7696, 7717, 7718, 7719, 7720, 7721, 7722,7723, 7724, 7725, 7726, 7727, 7728, 7729, 7730, 7731, 7732, 7733, 7734,7735, 7736, 7738, 7739, 7740, 7741, 7742, 7743, 7744, 7746, 7747, 7748,7749, 7750, 7751, 7752, 7757, 7758, 7759, 7760, 7761, 7762, 7763, 7764,7765, 7766, 7767, 7768, 7796, 7797, 7805, 7813, 7814, 7815, 7816, 7817,7818, 7819, 7820, 7821, 7822, 7823, 7824, 7825, 7826, 7827, 7828, 7829,7830, 7831, 7832, 7833, 7848, 7849, 7850, 7851, 7852, 7853, 7854, 7859,7860, 7861, 7862, 7863, 7864, 7865, 7866, 7867, 7869, 7870, 7874, 7875,7917, 7918, 7919, 7925, 7926, 7927, 7928, 7929, 7941, 7942, 7943, 7944,7945, 7946, 7947, 7948, 7949, 7950, 7951, 7952, 7953, 7955, 7956, 7957,7958, 7959, 7960, 7961, 7962, 7963, 7964, 7965, 7966, 7967, 7968, 7969,7970, 7971, 7972, 7974, 7975, 7976, 7977, 7978, 7979, 7980, 7981, 7982,7983, 7984, 7985, 7986, 7987, 7988, 7989, 7990, 7991, 7992, 7993, 7994,7995, 7996, 7997, 7998, 7999, 8000, 8011, 8012, 8013, 8014, 8015, 8016,8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027, 8028,8029, 8030, 8048, 8049, 8050, 8051, 8052, 8053, 8054, 8055, 8056, 8057,8087, 8088, 8089, 8090, 8091, 8092, 8095, 8096, 8097, 8098, 8099, 8100,8101, 8102, 8103, 8104, 8105, 8106, 8107, 8110, 8111, 8112, 8113, 8114,8115, 8116, 8117, 8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125, 8126,8127, 8130, 8131, 8132, 8133, 8134, 8135, 8136, 8144, 8145, 8151, 8152,8153, 8154, 8155, 8156, 8157, 8166, 8169, 8170, 8171, 8172, 8173, 8174,8175, 8176, 8177, 8178, 8179, 8180, 8181, 8182, 8188, 8191, 8192, 8193,8194, 8195, 8196, 8197, 8198, 8199, 8200, 8201, 8202, 8203, 8204, 8205,8206, 8207, 8208, 8209, 8210, 8211, 8212, 8215, 8221, 8227, 8230, 8231,8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241, 8242, 8243,8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251, 8260, 8261, 8262, 8263,8264, 8265, 8266, 8267, 8268, 8269, 8270, 8271, 8272, 8273, 8274, 8275,8276, 8277, 8278, 8279, 8280, 8281, 8282, 8283, 8284, 8299, 8302, 8303,8304, 8305, 8306, 8315, 8316, 8317, 8353, 8354, 8355, 8356, 8357, 8358,8359, 8360, 8361, 8375, 8376, 8377, 8378, 8379, 8380, 8381, 8382, 8383,8384, 8385, 8386, 8387, 8388, 8389, 8390, 8391, 8392, 8393, 8394, 8397,8398, 8399, 8400, 8401, 8402, 8403, 8404, 8405, 8406, 8410, 8411, 8412,8419, 8420, 8421, 8422, 8423, 8424, 8425, 8426, 8440, 8459, 8460, 8461,8462, 8463, 8473, 8474, 8475, 8476, 8477, 8478, 8508, 8509, 8510, 8511,8512, 8514, 8515, 8533, 8534, 8546, 8547, 8548, 8549, 8550, 8551, 8552,8553, 8554, 8555, 8570, 8571, 8572, 8573, 8574, 8575, 8576, 8577, 8578,8579, 8580, 8581, 8582, 8586, 8587, 8593, 8594, 8595, 8596, 8597, 8601,8602, 8603, 8604, 8605, 8610, 8611, 8612, 8613, 8614, 8615, 8617, 8618,8641, 8642, 8643, 8644, 8645, 8646, 8647, 8648, 8655, 8686, 8709, 8712,8713, 8714, 8715, 8716, 8717, 8718, 8719, 8720, 8721, 8722, 8723, 8724,8725, 8726, 8727, 8728, 8729, 8730, 8731, 8732, 8733, 8734, 8770, 8771,8788, 8789, 8790, 8791, 8792, 8793, 8794, 8795, 8796, 8797, 8798, 8799,8800, 8801, 8802, 8803, 8804, 8805, 8806, 8810, 8811, 8817, 8818, 8819,8820, 8821, 8822, 8823, 8829, 8830, 8837, 8838, 8839, 8840, 8841, 8848,8849, 8850, 8851, 8854, 8855, 8856, 8857, 8858, 8859, 8860, 8861, 8862,8863, 8864, 8865, 8867, 8868, 8872, 8873, 8874, 8875, 8876, 8878, 8879,8880, 8881, 8882, 8883, 8884, 8885, 8886, 8887, 8888, 8889, 8890, 8891,8892, 8893, 8894, 8895, 8896, 8897, 8898, 8899, 8900, 8901, 8902, 8903,8904, 8906, 8913, 8914, 8915, 8916, 8917, 8918, 8919, 8920, 8921, 8925,8926, 8927, 8928, 8933, 8934, 8935, 8936, 8937, 8938, 8939, 8940, 8941,8942, 8943, 8944, 8945, 8946, 8947, 8948, 8949, 8950, 8951, 8954, 8955,8956, 8992, 8993, 8994, 8995, 8996, 8997, 8998, 8999, 9000, 9001, 9002,9003, 9010, 9011, 9012, 9013, 9014, 9018, 9019, 9020, 9021, 9022, 9023,9024, 9025, 9026, 9027, 9028, 9029, 9030, 9031, 9041, 9042, 9043, 9044,9045, 9046, 9047, 9048, 9049, 9050, 9051, 9052, 9053, 9054, 9055, 9056,9057, 9058, 9059, 9065, 9066, 9087, 9088, 9089, 9090, 9091, 9092, 9093,9094, 9095, 9096, 9097, 9098, 9103, 9104, 9105, 9106, 9107, 9108, 9109,9110, 9123, 9124, 9125, 9126, 9127, 9128, 9129, 9130, 9131, 9133, 9134,9135, 9136, 9137, 9145, 9146, 9147, 9148, 9149, 9150, 9151, 9160, 9161,9162, 9163, 9201, 9202, 9203.

Liver Cancer

There are two types of liver cancer, primary liver cancer which forms inthe tissue of the liver and secondary liver cancer, or metastatic livercancer, that spreads to the liver from another part of the body.Possible symptoms of liver cancer include, but are not limited to, ahard lump on the right side just below the rib cage, discomfort in theupper abdomen on the right side, pain around the right shoulder blade,unexplained weight loss, jaundice, unusual tiredness, nausea and loss ofappetite.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withliver cancer by administering to said subject an isolated polynucleotideencoding an oncology-related polypeptide of interest. In one embodiment,the oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA of the present invention may beused to reduce, eliminate, or prevent tumor growth in a subject who hasbeen diagnosed or may be diagnosed with liver cancer by administering tosaid subject an isolated polynucleotide encoding an oncology-relatedpolypeptide of interest. In one embodiment, the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA of the present invention may be used to reduce and/or ameliorateat least one symptom of cancer in a subject who has been diagnosed ormay be diagnosed with liver cancer by administering to said subject anisolated polynucleotide encoding an oncology-related polypeptide ofinterest. In a further embodiment, the oncology-related polypeptide ofinterest may include, but not limited to, SEQ ID NOs: 4711, 4712, 4713,4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722, 4723, 4724, 4725,4726, 4727, 4728, 4742, 4743, 4744, 4745, 4746, 4747, 4748, 4749, 4750,4751, 4774, 4775, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789,4790, 4825, 4826, 4827, 4828, 4829, 4830, 4832, 4844, 4845, 4846, 4854,4855, 4856, 4857, 4858, 4859, 4860, 4861, 4863, 4864, 4865, 4866, 4867,4868, 4869, 4870, 4871, 4872, 4873, 4885, 4886, 4887, 4888, 4889, 4890,4891, 4892, 4893, 4894, 4911, 4912, 4913, 4914, 4915, 4916, 4917, 4918,4919, 4920, 4960, 4961, 4962, 4963, 4964, 4965, 4966, 4967, 4968, 4969,4970, 4971, 4972, 4973, 4974, 4975, 4976, 4977, 4978, 4979, 4980, 4981,4982, 4983, 4984, 4985, 4986, 4987, 4988, 4989, 4990, 4991, 4992, 4993,4994, 4995, 4996, 4997, 4998, 4999, 5000, 5001, 5002, 5008, 5009, 5010,5011, 5012, 5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021, 5022,5023, 5024, 5025, 5026, 5027, 5028, 5030, 5031, 5032, 5033, 5034, 5035,5036, 5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046, 5047,5048, 5049, 5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058, 5059,5060, 5061, 5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071,5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079, 5103, 5104, 5105, 5106,5107, 5108, 5109, 5110, 5118, 5119, 5120, 5121, 5122, 5123, 5124, 5125,5126, 5127, 5128, 5129, 5130, 5131, 5132, 5133, 5134, 5135, 5136, 5137,5138, 5139, 5140, 5141, 5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152,5153, 5154, 5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164,5165, 5166, 5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176,5177, 5178, 5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191,5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203,5204, 5205, 5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5214, 5215,5216, 5217, 5218, 5219, 5220, 5221, 5222, 5223, 5230, 5231, 5252, 5253,5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264, 5265,5266, 5267, 5268, 5269, 5270, 5271, 5291, 5292, 5293, 5294, 5295, 5296,5297, 5298, 5299, 5300, 5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308,5309, 5310, 5311, 5312, 5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320,5321, 5322, 5323, 5324, 5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332,5333, 5334, 5335, 5336, 5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344,5345, 5346, 5347, 5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356,5357, 5358, 5359, 5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368,5369, 5370, 5371, 5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380,5381, 5382, 5383, 5384, 5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392,5393, 5419, 5420, 5421, 5426, 5427, 5428, 5429, 5438, 5447, 5448, 5449,5450, 5451, 5452, 5453, 5454, 5455, 5456, 5457, 5458, 5459, 5460, 5461,5462, 5463, 5464, 5465, 5469, 5470, 5477, 5478, 5479, 5480, 5481, 5482,5490, 5491, 5492, 5493, 5494, 5495, 5496, 5497, 5498, 5499, 5504, 5505,5506, 5507, 5508, 5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516, 5517,5518, 5519, 5520, 5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529,5530, 5531, 5532, 5533, 5534, 5535, 5536, 5548, 5549, 5551, 5552, 5553,5554, 5555, 5556, 5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565,5566, 5567, 5568, 5569, 5570, 5571, 5575, 5576, 5577, 5578, 5579, 5580,5590, 5591, 5592, 5593, 5594, 5595, 5596, 5597, 5598, 5599, 5605, 5606,5607, 5608, 5609, 5610, 5611, 5616, 5617, 5618, 5619, 5620, 5621, 5622,5623, 5631, 5632, 5633, 5638, 5639, 5640, 5671, 5672, 5673, 5674, 5675,5676, 5677, 5678, 5679, 5680, 5681, 5688, 5689, 5690, 5691, 5692, 5693,5694, 5695, 5696, 5697, 5698, 5699, 5700, 5701, 5702, 5703, 5708, 5709,5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717, 5727, 5740, 5741, 5742,5743, 5744, 5745, 5746, 5747, 5748, 5749, 5750, 5751, 5755, 5757, 5758,5759, 5760, 5761, 5762, 5763, 5764, 5765, 5766, 5767, 5768, 5769, 5770,5772, 5773, 5775, 5776, 5777, 5778, 5779, 5780, 5783, 5784, 5785, 5786,5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796, 5797, 5798,5799, 5800, 5801, 5802, 5803, 5811, 5812, 5813, 5814, 5821, 5822, 5823,5824, 5825, 5826, 5827, 5828, 5829, 5830, 5831, 5833, 5834, 5836, 5837,5838, 5839, 5842, 5843, 5844, 5845, 5846, 5847, 5848, 5849, 5850, 5851,5852, 5853, 5854, 5855, 5864, 5865, 5866, 5867, 5868, 5875, 5876, 5877,5878, 5880, 5881, 5882, 5883, 5884, 5885, 5886, 5887, 5888, 5889, 5890,5891, 5892, 5893, 5894, 5895, 5896, 5897, 5898, 5899, 5907, 5908, 5909,5911, 5912, 5913, 5914, 5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922,5929, 5950, 5951, 5952, 5953, 5954, 5955, 5956, 5963, 5967, 5968, 5969,5970, 5982, 5983, 5984, 5985, 5986, 5987, 5988, 5997, 5998, 5999, 6000,6001, 6002, 6068, 6069, 6070, 6071, 6072, 6074, 6075, 6076, 6077, 6078,6080, 6081, 6082, 6083, 6084, 6085, 6086, 6087, 6088, 6089, 6090, 6091,6092, 6093, 6094, 6095, 6096, 6097, 6098, 6100, 6101, 6102, 6103, 6105,6106, 6107, 6108, 6109, 6110, 6111, 6112, 6113, 6114, 6115, 6116, 6117,6118, 6119, 6120, 6121, 6122, 6123, 6124, 6128, 6130, 6142, 6143, 6144,6145, 6146, 6147, 6148, 6149, 6151, 6152, 6153, 6154, 6155, 6156, 6157,6169, 6170, 6171, 6172, 6173, 6174, 6175, 6176, 6177, 6178, 6179, 6180,6181, 6182, 6185, 6186, 6187, 6191, 6208, 6209, 6231, 6232, 6233, 6234,6235, 6236, 6237, 6238, 6239, 6251, 6256, 6257, 6258, 6259, 6260, 6261,6262, 6278, 6279, 6280, 6281, 6282, 6286, 6287, 6288, 6289, 6290, 6291,6292, 6293, 6294, 6295, 6298, 6299, 6300, 6301, 6302, 6303, 6304, 6305,6306, 6307, 6308, 6309, 6310, 6311, 6312, 6313, 6314, 6315, 6356, 6357,6358, 6378, 6379, 6380, 6387, 6388, 6389, 6390, 6391, 6392, 6393, 6394,6395, 6396, 6397, 6398, 6399, 6400, 6401, 6402, 6403, 6405, 6406, 6407,6451, 6452, 6453, 6497, 6498, 6499, 6500, 6501, 6502, 6503, 6509, 6510,6511, 6512, 6514, 6519, 6520, 6521, 6522, 6523, 6549, 6550, 6551, 6552,6553, 6554, 6555, 6556, 6557, 6558, 6559, 6588, 6589, 6599, 6600, 6601,6602, 6603, 6604, 6605, 6606, 6611, 6612, 6613, 6614, 6615, 6616, 6617,6618, 6619, 6620, 6621, 6622, 6623, 6624, 6625, 6626, 6627, 6628, 6629,6630, 6631, 6632, 6633, 6634, 6638, 6639, 6640, 6641, 6642, 6643, 6644,6684, 6685, 6686, 6697, 6698, 6699, 6700, 6701, 6719, 6721, 6723, 6724,6754, 6755, 6756, 6757, 6758, 6759, 6760, 6761, 6762, 6763, 6764, 6796,6797, 6798, 6799, 6800, 6805, 6806, 6807, 6808, 6809, 6814, 6815, 6816,6817, 6818, 6824, 6825, 6826, 6831, 6832, 6833, 6834, 6844, 6845, 6846,6853, 6854, 6855, 6856, 6857, 6860, 6861, 6862, 6863, 6864, 6865, 6866,6867, 6868, 6869, 6872, 6873, 6875, 6876, 6877, 6878, 6879, 6885, 6888,6889, 6890, 6891, 6892, 6893, 6894, 6895, 6896, 6897, 6898, 6899, 6900,6901, 6902, 6903, 6904, 6905, 6906, 6907, 6908, 6909, 6910, 6911, 6912,6913, 6914, 6915, 6916, 6917, 6918, 6919, 6920, 6921, 6922, 6923, 6924,6925, 6926, 6927, 6928, 6929, 6930, 6931, 6932, 6933, 6934, 6935, 6936,6953, 6954, 6955, 6957, 6958, 6959, 6960, 6961, 6962, 6963, 6964, 6965,6966, 6967, 6968, 6969, 6970, 6971, 6972, 6973, 6983, 6984, 6985, 6988,6989, 6990, 6991, 6992, 6993, 6994, 6995, 7007, 7008, 7009, 7010, 7011,7012, 7015, 7016, 7017, 7018, 7019, 7020, 7021, 7034, 7035, 7084, 7086,7087, 7088, 7089, 7090, 7091, 7092, 7128, 7137, 7138, 7139, 7140, 7141,7142, 7143, 7144, 7145, 7146, 7147, 7148, 7149, 7150, 7151, 7152, 7153,7154, 7155, 7195, 7196, 7197, 7198, 7200, 7201, 7202, 7203, 7204, 7205,7206, 7207, 7208, 7209, 7210, 7211, 7212, 7213, 7214, 7215, 7216, 7217,7218, 7219, 7220, 7221, 7222, 7223, 7224, 7225, 7226, 7227, 7228, 7229,7230, 7231, 7232, 7233, 7234, 7235, 7236, 7237, 7238, 7239, 7240, 7241,7243, 7244, 7245, 7246, 7247, 7248, 7249, 7250, 7251, 7252, 7253, 7254,7255, 7256, 7257, 7258, 7259, 7260, 7261, 7262, 7263, 7264, 7265, 7266,7267, 7268, 7269, 7270, 7271, 7291, 7292, 7293, 7294, 7295, 7296, 7297,7298, 7347, 7348, 7349, 7358, 7359, 7360, 7361, 7362, 7363, 7364, 7365,7372, 7373, 7374, 7375, 7376, 7377, 7378, 7383, 7415, 7416, 7417, 7438,7471, 7472, 7473, 7474, 7475, 7476, 7477, 7490, 7492, 7493, 7494, 7495,7496, 7497, 7498, 7499, 7500, 7501, 7502, 7503, 7504, 7505, 7506, 7507,7508, 7509, 7510, 7511, 7512, 7513, 7514, 7515, 7532, 7533, 7534, 7535,7537, 7538, 7539, 7540, 7541, 7542, 7543, 7544, 7545, 7546, 7547, 7548,7549, 7550, 7551, 7552, 7553, 7554, 7555, 7556, 7557, 7558, 7559, 7560,7577, 7578, 7579, 7585, 7586, 7587, 7588, 7589, 7590, 7591, 7592, 7593,7595, 7596, 7597, 7599, 7600, 7601, 7617, 7618, 7619, 7620, 7621, 7622,7623, 7638, 7668, 7669, 7670, 7672, 7673, 7674, 7675, 7676, 7685, 7687,7694, 7695, 7696, 7717, 7718, 7719, 7720, 7721, 7722, 7723, 7724, 7725,7726, 7727, 7728, 7729, 7730, 7731, 7732, 7733, 7734, 7735, 7736, 7737,7738, 7739, 7740, 7741, 7742, 7743, 7744, 7746, 7747, 7748, 7749, 7750,7751, 7752, 7757, 7758, 7759, 7760, 7761, 7762, 7763, 7764, 7765, 7766,7767, 7768, 7796, 7797, 7805, 7813, 7814, 7815, 7816, 7817, 7818, 7819,7820, 7821, 7822, 7848, 7849, 7850, 7851, 7852, 7853, 7854, 7859, 7860,7861, 7862, 7863, 7864, 7865, 7866, 7867, 7869, 7870, 7874, 7875, 7917,7918, 7919, 7925, 7926, 7927, 7928, 7929, 7941, 7942, 7943, 7944, 7945,7946, 7947, 7948, 7949, 7950, 7951, 7952, 7953, 7955, 7956, 7957, 7958,7959, 7960, 7961, 7962, 7963, 7964, 7965, 7966, 7967, 7968, 7969, 7970,7971, 7972, 7974, 7975, 7976, 7977, 7978, 7979, 7980, 7981, 7982, 7983,7984, 7985, 7986, 7987, 7988, 7989, 7990, 7991, 7992, 7993, 7994, 7995,7996, 7997, 7998, 7999, 8000, 8011, 8012, 8013, 8014, 8015, 8016, 8017,8018, 8019, 8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027, 8028, 8029,8030, 8048, 8049, 8050, 8051, 8052, 8053, 8054, 8055, 8056, 8057, 8087,8088, 8089, 8090, 8091, 8092, 8095, 8096, 8097, 8098, 8099, 8100, 8101,8102, 8103, 8104, 8105, 8106, 8107, 8110, 8111, 8112, 8113, 8114, 8115,8116, 8117, 8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125, 8126, 8127,8130, 8131, 8132, 8133, 8134, 8135, 8136, 8144, 8145, 8146, 8147, 8148,8149, 8150, 8166, 8169, 8170, 8171, 8172, 8173, 8174, 8175, 8176, 8177,8178, 8179, 8180, 8181, 8182, 8188, 8191, 8192, 8193, 8194, 8195, 8196,8197, 8198, 8199, 8200, 8201, 8209, 8210, 8211, 8212, 8215, 8227, 8230,8231, 8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241, 8242,8243, 8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251, 8260, 8261, 8262,8263, 8299, 8302, 8303, 8304, 8305, 8306, 8353, 8354, 8355, 8356, 8357,8358, 8359, 8360, 8361, 8375, 8376, 8377, 8378, 8379, 8380, 8381, 8382,8410, 8411, 8412, 8440, 8459, 8460, 8461, 8462, 8463, 8464, 8465, 8466,8474, 8475, 8476, 8477, 8478, 8508, 8509, 8510, 8511, 8512, 8514, 8515,8546, 8547, 8548, 8549, 8550, 8551, 8552, 8553, 8554, 8555, 8570, 8571,8572, 8573, 8574, 8575, 8576, 8577, 8578, 8579, 8580, 8581, 8583, 8586,8587, 8593, 8594, 8595, 8596, 8597, 8601, 8602, 8603, 8604, 8605, 8615,8617, 8618, 8655, 8686, 8709, 8712, 8713, 8714, 8715, 8716, 8717, 8718,8719, 8720, 8721, 8722, 8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730,8731, 8732, 8733, 8734, 8735, 8736, 8770, 8771, 8788, 8789, 8790, 8791,8792, 8793, 8794, 8795, 8796, 8797, 8798, 8799, 8800, 8801, 8802, 8803,8804, 8805, 8806, 8810, 8811, 8817, 8818, 8819, 8820, 8821, 8822, 8823,8829, 8830, 8837, 8838, 8839, 8840, 8841, 8848, 8849, 8850, 8851, 8854,8855, 8856, 8857, 8858, 8859, 8860, 8861, 8862, 8863, 8864, 8865, 8867,8868, 8872, 8873, 8874, 8875, 8876, 8878, 8879, 8880, 8881, 8882, 8883,8884, 8885, 8886, 8887, 8888, 8889, 8890, 8891, 8892, 8893, 8894, 8895,8896, 8897, 8898, 8899, 8900, 8901, 8902, 8903, 8904, 8906, 8913, 8914,8915, 8916, 8917, 8918, 8919, 8920, 8921, 8925, 8926, 8927, 8928, 8933,8934, 8935, 8936, 8937, 8938, 8939, 8940, 8941, 8942, 8943, 8944, 8945,8946, 8947, 8948, 8949, 8950, 8951, 8954, 8955, 8956, 8992, 8993, 8994,8995, 8996, 8997, 8998, 8999, 9000, 9001, 9002, 9003, 9010, 9011, 9012,9013, 9014, 9018, 9019, 9020, 9021, 9022, 9023, 9024, 9025, 9026, 9027,9028, 9029, 9030, 9031, 9041, 9042, 9043, 9044, 9045, 9046, 9047, 9048,9049, 9050, 9051, 9052, 9053, 9054, 9055, 9056, 9057, 9058, 9059, 9065,9066, 9067, 9068, 9069, 9070, 9071, 9072, 9073, 9074, 9087, 9088, 9091,9092, 9093, 9094, 9095, 9096, 9097, 9098, 9103, 9104, 9105, 9106, 9107,9108, 9109, 9110, 9123, 9124, 9125, 9126, 9127, 9128, 9129, 9130, 9131,9133, 9134, 9135, 9136, 9137, 9145, 9146, 9147, 9148, 9149, 9150, 9151,9153, 9154, 9155, 9156, 9157, 9158, 9159, 9160, 9161, 9162, 9163, 9201,9203.

Lung Cancer

Lung cancer forms in the tissues of the lung usually in the cells liningthe air passages and is classified as either small cell lung cancer ornon-small cell lung cancer. There are two types of small cell lungcancer, small cell carcinoma and combined small cell carcinoma. Thetypes of on-small cell lung cancer are squamous cell carcinoma (cancerbegins in the squamous cells), large cell carcinoma (cancer may begin inseveral types of cells) and adenocarcinoma (cancer begins in the cellsthat line the alveoli and in cells that make mucus). Symptoms of lungcancer include, but are not limited to, chest discomfort or pain, coughthat does not go away or gets worse over time, trouble breathing,wheezing, blood in the sputum, hoarseness, loss of appetite, weight lossfor no known reason, feeling very tired, trouble swallowing and swellingin the face and/or veins in the neck.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withlung cancer by administering to said subject an isolated polynucleotideencoding an oncology-related polypeptide of interest. In one embodiment,the oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA of the present invention may beused to reduce, eliminate, or prevent tumor growth in a subject who hasbeen diagnosed or may be diagnosed with lung cancer by administering tosaid subject an isolated polynucleotide encoding an oncology-relatedpolypeptide of interest. In one embodiment, the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA of the present invention may be used to reduce and/or ameliorateat least one symptom of cancer in a subject who has been diagnosed ormay be diagnosed with lung cancer by administering to said subject anisolated polynucleotide encoding an oncology-related polypeptide ofinterest. In a further embodiment, the oncology-related polypeptide ofinterest may include, but not limited to, SEQ ID NOs: 4711, 4712, 4713,4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722, 4723, 4724, 4725,4726, 4727, 4728, 4742, 4743, 4744, 4745, 4746, 4747, 4748, 4749, 4750,4751, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791,4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4808,4825, 4826, 4827, 4828, 4829, 4830, 4831, 4832, 4851, 4852, 4853, 4854,4855, 4856, 4857, 4858, 4859, 4860, 4861, 4863, 4864, 4865, 4866, 4867,4868, 4869, 4870, 4871, 4872, 4873, 4885, 4886, 4887, 4888, 4889, 4890,4891, 4892, 4893, 4894, 4911, 4912, 4913, 4914, 4915, 4916, 4917, 4918,4919, 4920, 4960, 4961, 4962, 4963, 4964, 4965, 4966, 4967, 4968, 4969,4970, 4971, 4972, 4973, 4974, 4975, 4976, 4977, 4978, 4979, 4980, 4981,4982, 4983, 4984, 4985, 4986, 4987, 4988, 4989, 4990, 4991, 4992, 4993,4994, 4995, 4996, 4997, 4998, 4999, 5000, 5001, 5002, 5008, 5009, 5010,5011, 5012, 5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021, 5022,5023, 5024, 5025, 5026, 5027, 5028, 5030, 5031, 5032, 5033, 5034, 5035,5036, 5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046, 5047,5048, 5049, 5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058, 5059,5060, 5061, 5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071,5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079, 5103, 5104, 5105, 5106,5107, 5108, 5109, 5110, 5118, 5119, 5120, 5121, 5122, 5123, 5124, 5125,5126, 5127, 5128, 5129, 5130, 5131, 5132, 5133, 5134, 5135, 5136, 5137,5138, 5139, 5140, 5141, 5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152,5153, 5154, 5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164,5165, 5166, 5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176,5177, 5178, 5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191,5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203,5204, 5205, 5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5214, 5215,5216, 5217, 5218, 5219, 5220, 5221, 5222, 5223, 5230, 5231, 5252, 5253,5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264, 5265,5266, 5267, 5268, 5269, 5270, 5271, 5282, 5283, 5286, 5287, 5288, 5289,5291, 5292, 5293, 5294, 5295, 5296, 5297, 5298, 5299, 5300, 5301, 5302,5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311, 5312, 5313, 5314,5315, 5316, 5317, 5318, 5319, 5320, 5321, 5322, 5323, 5324, 5325, 5326,5327, 5328, 5329, 5330, 5331, 5332, 5333, 5334, 5335, 5336, 5337, 5338,5339, 5340, 5341, 5342, 5343, 5344, 5345, 5346, 5347, 5348, 5349, 5350,5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358, 5359, 5360, 5361, 5362,5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370, 5371, 5372, 5373, 5374,5375, 5376, 5377, 5378, 5379, 5380, 5381, 5382, 5383, 5384, 5385, 5386,5387, 5388, 5389, 5390, 5391, 5392, 5393, 5419, 5420, 5421, 5426, 5427,5428, 5429, 5438, 5447, 5448, 5449, 5450, 5451, 5452, 5453, 5454, 5455,5456, 5457, 5458, 5459, 5460, 5461, 5462, 5463, 5464, 5465, 5469, 5470,5477, 5478, 5479, 5480, 5481, 5482, 5490, 5491, 5492, 5493, 5494, 5495,5496, 5497, 5498, 5499, 5500, 5501, 5502, 5503, 5504, 5505, 5506, 5507,5508, 5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516, 5517, 5518, 5519,5520, 5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529, 5530, 5531,5532, 5533, 5534, 5535, 5536, 5548, 5549, 5551, 5552, 5553, 5554, 5555,5556, 5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565, 5566, 5567,5568, 5569, 5570, 5571, 5577, 5578, 5579, 5580, 5582, 5583, 5584, 5585,5586, 5587, 5590, 5591, 5592, 5593, 5594, 5595, 5596, 5600, 5601, 5602,5603, 5604, 5605, 5606, 5607, 5608, 5609, 5610, 5611, 5616, 5617, 5618,5619, 5620, 5621, 5622, 5623, 5624, 5625, 5626, 5631, 5632, 5633, 5638,5639, 5640, 5648, 5649, 5650, 5651, 5652, 5653, 5671, 5672, 5673, 5674,5675, 5676, 5677, 5678, 5679, 5680, 5681, 5688, 5689, 5690, 5691, 5692,5693, 5694, 5695, 5696, 5697, 5698, 5699, 5700, 5701, 5702, 5703, 5708,5709, 5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717, 5727, 5743, 5744,5745, 5746, 5747, 5748, 5749, 5750, 5751, 5755, 5757, 5758, 5759, 5760,5761, 5762, 5763, 5764, 5765, 5766, 5767, 5768, 5769, 5770, 5772, 5773,5775, 5776, 5777, 5778, 5779, 5780, 5783, 5784, 5785, 5786, 5787, 5788,5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796, 5797, 5798, 5799, 5800,5801, 5802, 5803, 5804, 5805, 5806, 5807, 5808, 5809, 5810, 5815, 5816,5817, 5821, 5822, 5823, 5824, 5825, 5826, 5827, 5828, 5829, 5830, 5831,5833, 5834, 5836, 5837, 5838, 5839, 5840, 5841, 5848, 5849, 5850, 5851,5852, 5853, 5854, 5855, 5864, 5865, 5866, 5867, 5868, 5875, 5876, 5877,5878, 5880, 5881, 5882, 5883, 5884, 5885, 5886, 5887, 5888, 5889, 5890,5891, 5892, 5893, 5894, 5895, 5896, 5897, 5898, 5899, 5906, 5907, 5908,5909, 5911, 5912, 5913, 5914, 5915, 5916, 5917, 5918, 5919, 5920, 5921,5922, 5929, 5950, 5951, 5952, 5953, 5954, 5955, 5956, 5963, 5964, 5965,5966, 5967, 5969, 5970, 5982, 5983, 5984, 5985, 5986, 5987, 5988, 5997,5998, 5999, 6000, 6001, 6002, 6029, 6068, 6069, 6070, 6071, 6072, 6074,6075, 6076, 6077, 6078, 6080, 6081, 6082, 6083, 6084, 6085, 6086, 6087,6088, 6089, 6090, 6091, 6092, 6093, 6094, 6095, 6096, 6097, 6098, 6100,6101, 6102, 6103, 6105, 6106, 6107, 6108, 6109, 6110, 6111, 6112, 6113,6114, 6115, 6116, 6117, 6118, 6119, 6120, 6121, 6122, 6123, 6124, 6128,6130, 6142, 6143, 6144, 6145, 6146, 6147, 6148, 6149, 6151, 6152, 6153,6154, 6155, 6156, 6157, 6169, 6170, 6171, 6172, 6173, 6174, 6175, 6176,6177, 6178, 6179, 6180, 6181, 6182, 6185, 6186, 6187, 6188, 6191, 6208,6209, 6231, 6232, 6233, 6234, 6235, 6236, 6237, 6238, 6239, 6251, 6256,6257, 6258, 6259, 6260, 6261, 6262, 6278, 6279, 6280, 6281, 6282, 6286,6287, 6288, 6289, 6290, 6298, 6299, 6300, 6301, 6302, 6303, 6304, 6305,6306, 6307, 6308, 6309, 6310, 6311, 6312, 6313, 6314, 6315, 6356, 6357,6358, 6360, 6361, 6362, 6363, 6364, 6375, 6376, 6377, 6378, 6379, 6387,6388, 6389, 6390, 6391, 6392, 6393, 6394, 6395, 6396, 6397, 6398, 6399,6400, 6401, 6402, 6403, 6405, 6406, 6407, 6451, 6452, 6453, 6497, 6498,6499, 6500, 6501, 6502, 6503, 6509, 6510, 6511, 6512, 6513, 6514, 6515,6516, 6517, 6518, 6519, 6520, 6521, 6522, 6523, 6549, 6550, 6551, 6552,6553, 6554, 6555, 6556, 6557, 6558, 6588, 6589, 6599, 6600, 6601, 6602,6603, 6604, 6605, 6606, 6611, 6612, 6613, 6614, 6615, 6616, 6617, 6618,6619, 6620, 6621, 6622, 6623, 6624, 6625, 6626, 6627, 6628, 6629, 6630,6631, 6632, 6633, 6634, 6638, 6639, 6640, 6641, 6642, 6643, 6644, 6645,6646, 6684, 6685, 6686, 6697, 6698, 6699, 6700, 6701, 6719, 6720, 6721,6723, 6724, 6754, 6755, 6756, 6757, 6758, 6759, 6760, 6761, 6762, 6763,6764, 6796, 6797, 6798, 6799, 6800, 6802, 6803, 6805, 6806, 6807, 6808,6809, 6814, 6815, 6816, 6817, 6818, 6824, 6825, 6826, 6831, 6832, 6833,6834, 6839, 6840, 6841, 6842, 6843, 6853, 6854, 6855, 6856, 6857, 6860,6861, 6862, 6863, 6864, 6865, 6866, 6867, 6868, 6869, 6872, 6873, 6874,6875, 6876, 6877, 6878, 6879, 6885, 6888, 6889, 6890, 6891, 6892, 6893,6894, 6895, 6896, 6897, 6898, 6899, 6900, 6901, 6902, 6903, 6904, 6905,6906, 6907, 6908, 6909, 6910, 6911, 6912, 6913, 6914, 6915, 6916, 6917,6918, 6919, 6920, 6921, 6922, 6923, 6924, 6925, 6926, 6927, 6928, 6929,6930, 6931, 6932, 6933, 6934, 6935, 6936, 6953, 6954, 6955, 6957, 6958,6959, 6960, 6962, 6963, 6964, 6965, 6966, 6967, 6968, 6969, 6970, 6971,6972, 6973, 6974, 6975, 6976, 6977, 6978, 6979, 6980, 6981, 6982, 6983,6984, 6985, 6988, 6989, 6990, 6991, 6992, 6993, 6994, 6995, 7007, 7008,7009, 7010, 7011, 7012, 7015, 7016, 7017, 7018, 7019, 7020, 7021, 7034,7035, 7084, 7085, 7086, 7087, 7088, 7089, 7090, 7091, 7092, 7128, 7137,7138, 7139, 7140, 7141, 7142, 7143, 7144, 7145, 7146, 7147, 7148, 7149,7150, 7151, 7152, 7153, 7154, 7155, 7195, 7196, 7197, 7198, 7200, 7201,7202, 7203, 7204, 7205, 7206, 7207, 7208, 7209, 7210, 7211, 7212, 7213,7214, 7215, 7216, 7217, 7218, 7219, 7220, 7221, 7222, 7223, 7224, 7225,7226, 7227, 7228, 7229, 7230, 7231, 7232, 7233, 7234, 7235, 7236, 7237,7238, 7239, 7240, 7241, 7243, 7244, 7245, 7246, 7247, 7248, 7249, 7250,7251, 7252, 7253, 7254, 7255, 7256, 7257, 7258, 7259, 7260, 7261, 7262,7263, 7264, 7265, 7266, 7267, 7268, 7269, 7270, 7271, 7291, 7292, 7293,7294, 7295, 7296, 7297, 7298, 7299, 7300, 7301, 7302, 7303, 7304, 7347,7348, 7349, 7350, 7351, 7352, 7353, 7354, 7358, 7359, 7360, 7361, 7362,7363, 7364, 7365, 7372, 7373, 7374, 7375, 7376, 7377, 7378, 7383, 7415,7416, 7417, 7438, 7471, 7472, 7473, 7474, 7475, 7476, 7477, 7490, 7496,7497, 7498, 7499, 7500, 7501, 7502, 7503, 7504, 7505, 7506, 7507, 7508,7509, 7510, 7511, 7512, 7513, 7514, 7515, 7516, 7517, 7518, 7519, 7520,7521, 7522, 7523, 7524, 7525, 7526, 7532, 7533, 7534, 7535, 7537, 7538,7539, 7540, 7541, 7542, 7543, 7544, 7545, 7546, 7547, 7548, 7549, 7550,7551, 7552, 7553, 7554, 7555, 7556, 7557, 7558, 7559, 7560, 7585, 7586,7587, 7588, 7589, 7590, 7591, 7592, 7593, 7595, 7596, 7597, 7599, 7600,7601, 7617, 7618, 7619, 7620, 7621, 7622, 7623, 7638, 7668, 7669, 7670,7672, 7673, 7674, 7675, 7676, 7685, 7687, 7694, 7695, 7696, 7717, 7718,7719, 7720, 7721, 7722, 7723, 7724, 7725, 7726, 7727, 7728, 7729, 7730,7731, 7732, 7733, 7734, 7735, 7736, 7737, 7738, 7739, 7740, 7741, 7742,7743, 7744, 7746, 7747, 7748, 7749, 7750, 7751, 7752, 7757, 7758, 7759,7760, 7761, 7762, 7763, 7764, 7765, 7766, 7767, 7768, 7796, 7797, 7805,7813, 7814, 7815, 7816, 7817, 7818, 7819, 7820, 7821, 7822, 7848, 7849,7850, 7851, 7852, 7853, 7854, 7859, 7860, 7861, 7862, 7863, 7864, 7865,7866, 7867, 7869, 7870, 7874, 7875, 7881, 7886, 7887, 7888, 7889, 7890,7891, 7892, 7893, 7917, 7918, 7919, 7925, 7926, 7927, 7928, 7929, 7941,7942, 7943, 7944, 7945, 7946, 7947, 7948, 7949, 7950, 7951, 7952, 7953,7955, 7956, 7957, 7958, 7959, 7960, 7961, 7962, 7963, 7964, 7965, 7966,7967, 7968, 7969, 7970, 7971, 7972, 7974, 7975, 7976, 7977, 7978, 7979,7980, 7981, 7982, 7983, 7984, 7985, 7986, 7987, 7988, 7989, 7990, 7991,7992, 7993, 7994, 7995, 7996, 7997, 7998, 7999, 8000, 8011, 8012, 8013,8014, 8015, 8016, 8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024, 8025,8026, 8027, 8028, 8029, 8030, 8048, 8049, 8050, 8051, 8052, 8053, 8054,8055, 8056, 8057, 8087, 8088, 8089, 8090, 8091, 8092, 8093, 8094, 8095,8096, 8097, 8098, 8099, 8100, 8101, 8102, 8103, 8104, 8105, 8106, 8107,8110, 8111, 8112, 8113, 8114, 8115, 8116, 8117, 8118, 8119, 8120, 8121,8122, 8123, 8124, 8125, 8126, 8127, 8130, 8131, 8132, 8133, 8134, 8135,8136, 8144, 8145, 8166, 8169, 8170, 8171, 8172, 8173, 8174, 8175, 8176,8177, 8178, 8179, 8180, 8181, 8182, 8188, 8191, 8192, 8193, 8194, 8195,8196, 8197, 8198, 8199, 8200, 8201, 8209, 8210, 8211, 8212, 8215, 8227,8230, 8231, 8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241,8242, 8243, 8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251, 8260, 8261,8262, 8263, 8299, 8302, 8303, 8304, 8305, 8306, 8307, 8308, 8326, 8353,8354, 8355, 8356, 8357, 8358, 8359, 8360, 8361, 8375, 8376, 8377, 8378,8379, 8380, 8381, 8382, 8407, 8408, 8409, 8410, 8411, 8412, 8440, 8459,8460, 8461, 8462, 8463, 8467, 8468, 8474, 8475, 8476, 8477, 8478, 8508,8509, 8510, 8511, 8512, 8514, 8515, 8546, 8547, 8548, 8549, 8550, 8551,8552, 8553, 8554, 8555, 8570, 8571, 8572, 8573, 8574, 8575, 8576, 8577,8578, 8579, 8580, 8581, 8586, 8587, 8593, 8594, 8595, 8596, 8597, 8598,8599, 8600, 8601, 8602, 8603, 8604, 8605, 8615, 8617, 8618, 8655, 8686,8709, 8712, 8713, 8714, 8715, 8716, 8717, 8718, 8719, 8720, 8721, 8722,8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730, 8731, 8732, 8733, 8734,8768, 8769, 8770, 8771, 8772, 8773, 8788, 8789, 8790, 8791, 8792, 8793,8794, 8795, 8796, 8797, 8798, 8799, 8800, 8801, 8802, 8803, 8804, 8805,8806, 8810, 8811, 8817, 8818, 8819, 8820, 8821, 8822, 8823, 8829, 8830,8837, 8838, 8839, 8840, 8841, 8848, 8849, 8850, 8851, 8854, 8855, 8856,8857, 8858, 8859, 8860, 8861, 8862, 8863, 8864, 8865, 8867, 8868, 8872,8873, 8874, 8875, 8876, 8878, 8879, 8880, 8881, 8882, 8883, 8884, 8885,8886, 8887, 8888, 8889, 8890, 8891, 8892, 8893, 8894, 8895, 8896, 8897,8898, 8899, 8900, 8901, 8902, 8903, 8904, 8906, 8913, 8914, 8915, 8916,8917, 8918, 8919, 8920, 8921, 8925, 8926, 8927, 8928, 8933, 8934, 8935,8936, 8937, 8938, 8939, 8940, 8941, 8942, 8943, 8944, 8945, 8946, 8947,8948, 8949, 8950, 8951, 8954, 8955, 8956, 8992, 8993, 8994, 8995, 8996,8997, 8998, 8999, 9000, 9001, 9002, 9003, 9010, 9011, 9012, 9013, 9014,9018, 9019, 9020, 9021, 9022, 9023, 9024, 9025, 9026, 9027, 9028, 9029,9030, 9031, 9041, 9042, 9043, 9044, 9045, 9046, 9047, 9048, 9049, 9050,9051, 9052, 9053, 9054, 9055, 9056, 9057, 9058, 9059, 9065, 9066, 9087,9088, 9091, 9092, 9093, 9094, 9095, 9096, 9097, 9098, 9103, 9104, 9105,9106, 9107, 9108, 9109, 9110, 9123, 9124, 9125, 9126, 9127, 9128, 9129,9130, 9131, 9133, 9134, 9135, 9136, 9137, 9145, 9146, 9147, 9148, 9149,9150, 9151, 9160, 9161, 9162, 9163, 9201, 9203.

Lymphoma

Lymphoma is cancer that beings in the cells of the immune system.Subjects who have Hodgkin lymphoma have a cell called Reed-Sternbergcell and non-Hodgkin lymphoma includes a large group of cancers ofimmune system cells. Examples of Lymphoma include, but are not limitedto, painless, swollen lymph nodes in the neck, underarm or groin, feverfor no known reason, drenching night sweats, weight loss for no knownreason, itchy skin and fatigue.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withlymphoma by administering to said subject an isolated polynucleotideencoding an oncology-related polypeptide of interest. In one embodiment,the oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA of the present invention may beused to reduce, eliminate, or prevent tumor growth in a subject who hasbeen diagnosed or may be diagnosed with lymphoma by administering tosaid subject an isolated polynucleotide encoding an oncology-relatedpolypeptide of interest. In one embodiment, the oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA of the present invention may be used to reduce and/or ameliorateat least one symptom of cancer in a subject who has been diagnosed ormay be diagnosed with lymphoma by administering to said subject anisolated polynucleotide encoding an oncology-related polypeptide ofinterest. In a further embodiment, the oncology-related polypeptide ofinterest may include, but not limited to, SEQ ID NOs: 4711, 4712, 4713,4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722, 4723, 4724, 4725,4726, 4727, 4728, 4742, 4743, 4744, 4745, 4746, 4747, 4748, 4749, 4750,4751, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4809,4810, 4811, 4812, 4813, 4814, 4825, 4826, 4827, 4828, 4829, 4830, 4832,4833, 4834, 4850, 4854, 4855, 4856, 4857, 4858, 4859, 4860, 4861, 4863,4864, 4865, 4866, 4867, 4868, 4869, 4870, 4871, 4872, 4873, 4885, 4886,4887, 4888, 4889, 4890, 4891, 4892, 4893, 4894, 4911, 4912, 4913, 4914,4915, 4916, 4917, 4918, 4919, 4920, 4960, 4961, 4962, 4963, 4964, 4965,4966, 4967, 4968, 4969, 4970, 4971, 4972, 4973, 4974, 4975, 4976, 4977,4978, 4979, 4980, 4981, 4982, 4983, 4984, 4985, 4986, 4987, 4988, 4989,4990, 4991, 4992, 4993, 4994, 4995, 4996, 4997, 4998, 4999, 5000, 5001,5002, 5003, 5004, 5005, 5006, 5007, 5008, 5009, 5010, 5011, 5012, 5013,5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021, 5022, 5023, 5024, 5025,5026, 5027, 5028, 5030, 5031, 5032, 5033, 5034, 5035, 5036, 5037, 5038,5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046, 5047, 5048, 5049, 5050,5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058, 5059, 5060, 5061, 5062,5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071, 5072, 5073, 5074,5075, 5076, 5077, 5078, 5079, 5103, 5104, 5105, 5106, 5107, 5108, 5109,5110, 5118, 5119, 5120, 5121, 5122, 5123, 5124, 5125, 5126, 5127, 5128,5129, 5130, 5131, 5132, 5133, 5134, 5135, 5136, 5137, 5138, 5139, 5140,5141, 5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152, 5153, 5154, 5155,5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164, 5165, 5166, 5167,5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176, 5177, 5178, 5182,5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191, 5192, 5193, 5194,5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203, 5204, 5205, 5206,5207, 5208, 5209, 5210, 5211, 5212, 5213, 5214, 5215, 5216, 5217, 5218,5219, 5220, 5221, 5222, 5223, 5230, 5231, 5252, 5253, 5254, 5255, 5256,5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264, 5265, 5266, 5267, 5268,5269, 5270, 5271, 5291, 5292, 5293, 5294, 5295, 5296, 5297, 5298, 5299,5300, 5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311,5312, 5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320, 5321, 5322, 5323,5324, 5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332, 5333, 5334, 5335,5336, 5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344, 5345, 5346, 5347,5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358, 5359,5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370, 5371,5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380, 5381, 5382, 5383,5384, 5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392, 5393, 5419, 5420,5421, 5426, 5427, 5428, 5429, 5430, 5431, 5432, 5433, 5434, 5435, 5436,5437, 5438, 5439, 5440, 5441, 5442, 5443, 5444, 5445, 5446, 5447, 5448,5449, 5450, 5451, 5452, 5453, 5454, 5455, 5456, 5457, 5458, 5459, 5460,5461, 5462, 5463, 5464, 5465, 5466, 5467, 5468, 5469, 5470, 5471, 5472,5473, 5474, 5475, 5476, 5477, 5478, 5479, 5480, 5481, 5482, 5483, 5484,5485, 5486, 5487, 5488, 5489, 5490, 5491, 5492, 5493, 5494, 5495, 5496,5497, 5498, 5499, 5500, 5501, 5502, 5503, 5504, 5505, 5506, 5507, 5508,5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516, 5517, 5518, 5519, 5520,5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529, 5530, 5531, 5532,5533, 5534, 5535, 5536, 5548, 5549, 5551, 5552, 5553, 5554, 5555, 5556,5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565, 5566, 5567, 5568,5569, 5570, 5571, 5577, 5578, 5579, 5580, 5590, 5591, 5592, 5593, 5594,5595, 5596, 5605, 5606, 5607, 5608, 5609, 5610, 5611, 5612, 5613, 5614,5615, 5616, 5617, 5618, 5619, 5620, 5621, 5622, 5623, 5631, 5632, 5633,5638, 5639, 5640, 5666, 5667, 5668, 5669, 5671, 5672, 5673, 5674, 5675,5676, 5677, 5678, 5679, 5680, 5681, 5688, 5689, 5690, 5691, 5692, 5693,5694, 5695, 5696, 5697, 5698, 5699, 5700, 5701, 5702, 5703, 5708, 5709,5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717, 5727, 5740, 5741, 5743,5744, 5745, 5746, 5747, 5748, 5749, 5750, 5751, 5755, 5757, 5758, 5759,5760, 5761, 5762, 5763, 5764, 5765, 5766, 5767, 5768, 5769, 5770, 5772,5773, 5775, 5776, 5777, 5778, 5779, 5780, 5783, 5784, 5785, 5786, 5787,5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796, 5797, 5798, 5799,5800, 5801, 5802, 5803, 5804, 5805, 5806, 5807, 5808, 5809, 5810, 5811,5812, 5813, 5814, 5815, 5816, 5817, 5818, 5819, 5820, 5821, 5822, 5823,5824, 5825, 5826, 5827, 5828, 5829, 5830, 5831, 5833, 5834, 5836, 5837,5838, 5839, 5848, 5849, 5850, 5851, 5852, 5853, 5854, 5855, 5864, 5865,5866, 5867, 5868, 5875, 5876, 5877, 5878, 5880, 5881, 5882, 5883, 5884,5885, 5886, 5887, 5888, 5889, 5890, 5891, 5892, 5893, 5894, 5895, 5896,5897, 5898, 5899, 5900, 5901, 5907, 5908, 5909, 5911, 5912, 5913, 5914,5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922, 5929, 5950, 5951, 5952,5953, 5954, 5955, 5956, 5963, 5967, 5969, 5970, 5982, 5983, 5984, 5985,5986, 5987, 5988, 5997, 5998, 5999, 6000, 6001, 6002, 6068, 6069, 6070,6071, 6072, 6074, 6075, 6076, 6077, 6078, 6080, 6081, 6082, 6083, 6084,6085, 6086, 6087, 6088, 6089, 6090, 6091, 6092, 6093, 6094, 6095, 6096,6097, 6098, 6100, 6101, 6102, 6103, 6105, 6106, 6107, 6108, 6109, 6110,6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118, 6119, 6120, 6121, 6122,6123, 6124, 6128, 6130, 6142, 6143, 6144, 6145, 6146, 6147, 6148, 6149,6151, 6152, 6153, 6154, 6155, 6156, 6157, 6169, 6170, 6171, 6172, 6173,6174, 6175, 6176, 6177, 6178, 6179, 6180, 6181, 6182, 6185, 6186, 6187,6191, 6192, 6193, 6194, 6195, 6208, 6209, 6231, 6232, 6233, 6234, 6235,6236, 6237, 6238, 6239, 6251, 6256, 6257, 6258, 6259, 6260, 6261, 6262,6271, 6272, 6273, 6278, 6279, 6280, 6281, 6282, 6286, 6287, 6288, 6289,6290, 6298, 6299, 6300, 6301, 6302, 6303, 6304, 6305, 6306, 6307, 6308,6309, 6310, 6311, 6312, 6313, 6314, 6315, 6356, 6357, 6358, 6378, 6379,6387, 6388, 6389, 6390, 6391, 6392, 6393, 6394, 6395, 6396, 6397, 6398,6399, 6400, 6401, 6402, 6403, 6405, 6406, 6407, 6451, 6452, 6453, 6497,6498, 6499, 6500, 6501, 6502, 6503, 6509, 6510, 6511, 6512, 6514, 6519,6520, 6521, 6522, 6523, 6549, 6550, 6551, 6552, 6553, 6554, 6555, 6556,6557, 6558, 6588, 6589, 6599, 6600, 6601, 6602, 6603, 6604, 6605, 6606,6611, 6612, 6613, 6614, 6615, 6616, 6617, 6618, 6619, 6620, 6621, 6622,6623, 6624, 6625, 6626, 6627, 6628, 6629, 6630, 6631, 6632, 6633, 6634,6638, 6639, 6640, 6641, 6642, 6643, 6644, 6684, 6685, 6686, 6687, 6697,6698, 6699, 6700, 6701, 6719, 6721, 6723, 6724, 6754, 6755, 6756, 6757,6758, 6759, 6760, 6761, 6762, 6763, 6764, 6796, 6797, 6798, 6799, 6800,6805, 6806, 6807, 6808, 6809, 6814, 6815, 6816, 6817, 6818, 6824, 6825,6826, 6831, 6832, 6833, 6834, 6835, 6836, 6837, 6848, 6849, 6850, 6851,6852, 6853, 6854, 6855, 6856, 6857, 6860, 6861, 6862, 6863, 6864, 6865,6866, 6867, 6868, 6869, 6872, 6873, 6875, 6876, 6877, 6878, 6879, 6885,6888, 6889, 6890, 6891, 6892, 6893, 6894, 6895, 6896, 6897, 6898, 6899,6900, 6901, 6902, 6903, 6904, 6905, 6906, 6907, 6908, 6909, 6910, 6911,6912, 6913, 6914, 6915, 6916, 6917, 6918, 6919, 6920, 6921, 6922, 6923,6924, 6925, 6926, 6927, 6928, 6929, 6930, 6931, 6932, 6933, 6934, 6935,6936, 6937, 6938, 6939, 6940, 6941, 6942, 6943, 6944, 6945, 6946, 6947,6948, 6949, 6950, 6951, 6952, 6953, 6954, 6955, 6957, 6958, 6959, 6960,6962, 6963, 6964, 6965, 6966, 6967, 6968, 6969, 6970, 6971, 6972, 6973,6983, 6984, 6985, 6988, 6989, 6990, 6991, 6992, 6993, 6994, 6995, 7007,7008, 7009, 7010, 7011, 7012, 7015, 7016, 7017, 7018, 7019, 7020, 7021,7034, 7035, 7084, 7086, 7087, 7088, 7089, 7090, 7091, 7092, 7128, 7137,7138, 7139, 7140, 7141, 7142, 7143, 7144, 7145, 7146, 7147, 7148, 7149,7150, 7151, 7152, 7153, 7154, 7155, 7195, 7196, 7197, 7198, 7199, 7200,7201, 7202, 7203, 7204, 7205, 7206, 7207, 7208, 7209, 7210, 7211, 7212,7213, 7214, 7215, 7216, 7217, 7218, 7219, 7220, 7221, 7222, 7223, 7224,7225, 7226, 7227, 7228, 7229, 7230, 7231, 7232, 7233, 7234, 7235, 7236,7237, 7238, 7239, 7240, 7241, 7243, 7244, 7245, 7246, 7247, 7248, 7249,7250, 7251, 7252, 7253, 7254, 7255, 7256, 7257, 7258, 7259, 7260, 7261,7262, 7263, 7264, 7265, 7266, 7267, 7268, 7269, 7270, 7271, 7287, 7288,7289, 7291, 7292, 7293, 7294, 7295, 7296, 7297, 7298, 7347, 7348, 7349,7358, 7359, 7360, 7361, 7362, 7363, 7364, 7365, 7372, 7373, 7374, 7375,7376, 7377, 7378, 7383, 7415, 7416, 7417, 7438, 7471, 7472, 7473, 7474,7475, 7476, 7477, 7490, 7496, 7497, 7498, 7499, 7500, 7501, 7502, 7503,7504, 7505, 7506, 7507, 7508, 7509, 7510, 7511, 7512, 7513, 7514, 7515,7532, 7533, 7534, 7535, 7537, 7538, 7539, 7540, 7541, 7542, 7543, 7544,7545, 7546, 7547, 7548, 7549, 7550, 7551, 7552, 7553, 7554, 7555, 7556,7557, 7558, 7559, 7560, 7571, 7572, 7573, 7574, 7575, 7576, 7585, 7586,7587, 7588, 7589, 7590, 7591, 7592, 7593, 7595, 7596, 7597, 7599, 7600,7601, 7617, 7618, 7619, 7620, 7621, 7622, 7623, 7638, 7668, 7669, 7670,7672, 7673, 7674, 7675, 7676, 7677, 7685, 7687, 7689, 7694, 7695, 7696,7717, 7718, 7719, 7720, 7721, 7722, 7723, 7724, 7725, 7726, 7727, 7728,7729, 7730, 7731, 7732, 7733, 7734, 7735, 7736, 7737, 7738, 7739, 7740,7741, 7742, 7743, 7744, 7746, 7747, 7748, 7749, 7750, 7751, 7752, 7757,7758, 7759, 7760, 7761, 7762, 7763, 7764, 7765, 7766, 7767, 7768, 7796,7797, 7805, 7813, 7814, 7815, 7816, 7817, 7818, 7819, 7820, 7821, 7822,7848, 7849, 7850, 7851, 7852, 7853, 7854, 7859, 7860, 7861, 7862, 7863,7864, 7865, 7866, 7867, 7869, 7870, 7874, 7875, 7917, 7918, 7919, 7920,7921, 7925, 7926, 7927, 7928, 7929, 7930, 7931, 7932, 7933, 7934, 7935,7936, 7937, 7938, 7939, 7940, 7941, 7942, 7943, 7944, 7945, 7946, 7947,7948, 7949, 7950, 7951, 7952, 7953, 7955, 7956, 7957, 7958, 7959, 7960,7961, 7962, 7963, 7964, 7965, 7966, 7967, 7968, 7969, 7970, 7971, 7972,7974, 7975, 7976, 7977, 7978, 7979, 7980, 7981, 7982, 7983, 7984, 7985,7986, 7987, 7988, 7989, 7990, 7991, 7992, 7993, 7994, 7995, 7996, 7997,7998, 7999, 8000, 8011, 8012, 8013, 8014, 8015, 8016, 8017, 8018, 8019,8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027, 8028, 8029, 8030, 8048,8049, 8050, 8051, 8052, 8053, 8054, 8055, 8056, 8057, 8087, 8088, 8089,8090, 8091, 8092, 8095, 8096, 8097, 8098, 8099, 8100, 8101, 8102, 8103,8104, 8105, 8106, 8107, 8110, 8111, 8112, 8113, 8114, 8115, 8116, 8117,8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125, 8126, 8127, 8130, 8131,8132, 8133, 8134, 8135, 8136, 8144, 8145, 8166, 8169, 8170, 8171, 8172,8173, 8174, 8175, 8176, 8177, 8178, 8179, 8180, 8181, 8182, 8188, 8189,8190, 8191, 8192, 8193, 8194, 8195, 8196, 8197, 8198, 8199, 8200, 8201,8209, 8210, 8211, 8212, 8215, 8227, 8230, 8231, 8232, 8233, 8234, 8235,8236, 8237, 8238, 8239, 8240, 8241, 8242, 8243, 8244, 8245, 8246, 8247,8248, 8249, 8250, 8251, 8260, 8261, 8262, 8263, 8299, 8302, 8303, 8304,8305, 8306, 8353, 8354, 8355, 8356, 8357, 8358, 8359, 8360, 8361, 8375,8376, 8377, 8378, 8379, 8380, 8381, 8382, 8410, 8411, 8412, 8414, 8415,8416, 8417, 8418, 8440, 8459, 8460, 8461, 8462, 8463, 8474, 8475, 8476,8477, 8478, 8487, 8488, 8489, 8490, 8491, 8492, 8493, 8494, 8508, 8509,8510, 8511, 8512, 8514, 8515, 8546, 8547, 8548, 8549, 8550, 8551, 8552,8553, 8554, 8555, 8570, 8571, 8572, 8573, 8574, 8575, 8576, 8577, 8578,8579, 8580, 8581, 8586, 8587, 8593, 8594, 8595, 8596, 8597, 8601, 8602,8603, 8604, 8605, 8615, 8617, 8618, 8652, 8655, 8686, 8709, 8712, 8713,8714, 8715, 8716, 8717, 8718, 8719, 8720, 8721, 8722, 8723, 8724, 8725,8726, 8727, 8728, 8729, 8730, 8731, 8732, 8733, 8734, 8737, 8738, 8739,8740, 8770, 8771, 8788, 8789, 8790, 8791, 8792, 8793, 8794, 8795, 8796,8797, 8798, 8799, 8800, 8801, 8802, 8803, 8804, 8805, 8806, 8810, 8811,8817, 8818, 8819, 8820, 8821, 8822, 8823, 8829, 8830, 8837, 8838, 8839,8840, 8841, 8848, 8849, 8850, 8851, 8854, 8855, 8856, 8857, 8858, 8859,8860, 8861, 8862, 8863, 8864, 8865, 8867, 8868, 8869, 8870, 8871, 8872,8873, 8874, 8875, 8876, 8878, 8879, 8880, 8881, 8882, 8883, 8884, 8885,8886, 8887, 8888, 8889, 8890, 8891, 8892, 8893, 8894, 8895, 8896, 8897,8898, 8899, 8900, 8901, 8902, 8903, 8904, 8906, 8913, 8914, 8915, 8916,8917, 8918, 8919, 8920, 8921, 8925, 8926, 8927, 8928, 8933, 8934, 8935,8936, 8937, 8938, 8939, 8940, 8941, 8942, 8943, 8944, 8945, 8946, 8947,8948, 8949, 8950, 8951, 8954, 8955, 8956, 8992, 8993, 8994, 8995, 8996,8997, 8998, 8999, 9000, 9001, 9002, 9003, 9010, 9011, 9012, 9013, 9014,9015, 9016, 9017, 9018, 9019, 9020, 9021, 9022, 9023, 9024, 9025, 9026,9027, 9028, 9029, 9030, 9031, 9041, 9042, 9043, 9044, 9045, 9046, 9047,9048, 9049, 9050, 9051, 9052, 9053, 9054, 9055, 9056, 9057, 9058, 9059,9065, 9066, 9087, 9088, 9091, 9092, 9093, 9094, 9095, 9096, 9097, 9098,9103, 9104, 9105, 9106, 9107, 9108, 9109, 9110, 9123, 9124, 9125, 9126,9127, 9128, 9129, 9130, 9131, 9133, 9134, 9135, 9136, 9137, 9145, 9146,9147, 9148, 9149, 9150, 9151, 9160, 9161, 9162, 9163, 9201, 9203.

Ovarian Cancer

Ovarian cancer is cancer which forms in the tissues of the ovary whichare either ovarian epithelial carcinomas (begins on the surface of theovary) or malignant germ cell tumors (cancer that begins in the eggcells). Symptoms of ovarian cancer include, but are not limited to, painor swelling in the abdomen, pain in the pelvis, gastrointestinalproblems such as gas, bloating, or constipation and vaginal bleedingafter menopause.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withovarian cancer by administering to said subject an isolatedpolynucleotide encoding an oncology-related polypeptide of interest. Inone embodiment, the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA of the present inventionmay be used to reduce, eliminate, or prevent tumor growth in a subjectwho has been diagnosed or may be diagnosed with ovarian cancer byadministering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In one embodiment, theoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA of the present invention may be used to reduceand/or ameliorate at least one symptom of cancer in a subject who hasbeen diagnosed or may be diagnosed with ovarian cancer by administeringto said subject an isolated polynucleotide encoding an oncology-relatedpolypeptide of interest. In a further embodiment, the oncology-relatedpolypeptide of interest may include, but not limited to, SEQ ID NOs:4711, 4712, 4713, 4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722,4723, 4724, 4725, 4726, 4727, 4728, 4742, 4743, 4744, 4745, 4746, 4747,4748, 4749, 4750, 4751, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788,4789, 4790, 4825, 4826, 4827, 4828, 4829, 4830, 4832, 4854, 4855, 4856,4857, 4858, 4859, 4860, 4861, 4863, 4864, 4865, 4866, 4867, 4868, 4869,4870, 4871, 4872, 4873, 4885, 4886, 4887, 4888, 4889, 4890, 4891, 4892,4893, 4894, 4911, 4912, 4913, 4914, 4915, 4916, 4917, 4918, 4919, 4920,4960, 4961, 4962, 4963, 4964, 4965, 4966, 4967, 4968, 4969, 4970, 4971,4972, 4973, 4974, 4975, 4976, 4977, 4978, 4979, 4980, 4981, 4982, 4983,4984, 4985, 4986, 4987, 4988, 4989, 4990, 4991, 4992, 4993, 4994, 4995,4996, 4997, 4998, 4999, 5000, 5001, 5002, 5008, 5009, 5010, 5011, 5012,5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021, 5022, 5023, 5024,5025, 5026, 5027, 5028, 5030, 5031, 5032, 5033, 5034, 5035, 5036, 5037,5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046, 5047, 5048, 5049,5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058, 5059, 5060, 5061,5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071, 5072, 5073,5074, 5075, 5076, 5077, 5078, 5079, 5103, 5104, 5105, 5106, 5107, 5108,5109, 5110, 5118, 5119, 5120, 5121, 5122, 5123, 5124, 5125, 5126, 5127,5128, 5129, 5130, 5131, 5132, 5133, 5134, 5135, 5136, 5137, 5138, 5139,5140, 5141, 5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152, 5153, 5154,5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164, 5165, 5166,5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176, 5177, 5178,5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191, 5192, 5193,5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203, 5204, 5205,5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5230, 5231, 5252, 5253,5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264, 5265,5266, 5267, 5268, 5269, 5270, 5271, 5291, 5292, 5293, 5294, 5295, 5296,5297, 5298, 5299, 5300, 5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308,5309, 5310, 5311, 5312, 5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320,5321, 5322, 5323, 5324, 5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332,5333, 5334, 5335, 5336, 5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344,5345, 5346, 5347, 5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356,5357, 5358, 5359, 5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368,5369, 5370, 5371, 5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380,5381, 5382, 5383, 5384, 5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392,5393, 5419, 5420, 5421, 5426, 5427, 5428, 5429, 5438, 5447, 5448, 5449,5450, 5451, 5452, 5453, 5454, 5455, 5456, 5457, 5458, 5459, 5460, 5461,5462, 5463, 5464, 5465, 5469, 5470, 5477, 5478, 5479, 5480, 5481, 5482,5490, 5491, 5492, 5493, 5494, 5495, 5496, 5497, 5498, 5499, 5504, 5505,5506, 5507, 5508, 5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516, 5517,5518, 5519, 5520, 5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529,5530, 5531, 5532, 5533, 5534, 5535, 5536, 5548, 5549, 5551, 5552, 5553,5554, 5555, 5556, 5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565,5566, 5567, 5568, 5569, 5570, 5571, 5577, 5578, 5579, 5580, 5590, 5591,5592, 5593, 5594, 5595, 5596, 5605, 5606, 5607, 5608, 5609, 5610, 5611,5616, 5617, 5618, 5619, 5620, 5621, 5622, 5623, 5631, 5632, 5633, 5638,5639, 5640, 5671, 5672, 5673, 5674, 5675, 5676, 5677, 5678, 5679, 5680,5681, 5688, 5689, 5690, 5691, 5692, 5693, 5694, 5695, 5696, 5697, 5698,5699, 5700, 5701, 5702, 5703, 5708, 5709, 5710, 5711, 5712, 5713, 5714,5715, 5716, 5717, 5727, 5740, 5741, 5743, 5744, 5745, 5746, 5747, 5748,5749, 5750, 5751, 5755, 5757, 5758, 5759, 5760, 5761, 5762, 5763, 5764,5765, 5766, 5767, 5768, 5769, 5770, 5772, 5773, 5775, 5776, 5777, 5778,5779, 5780, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792,5793, 5794, 5795, 5796, 5797, 5798, 5799, 5800, 5801, 5802, 5803, 5811,5812, 5813, 5814, 5821, 5822, 5823, 5824, 5825, 5826, 5827, 5828, 5829,5830, 5831, 5833, 5834, 5836, 5837, 5838, 5839, 5848, 5849, 5850, 5851,5852, 5853, 5854, 5855, 5864, 5865, 5866, 5867, 5868, 5875, 5876, 5877,5878, 5880, 5881, 5882, 5883, 5884, 5885, 5886, 5887, 5888, 5889, 5890,5891, 5892, 5893, 5894, 5895, 5896, 5897, 5898, 5899, 5907, 5908, 5909,5911, 5912, 5913, 5914, 5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922,5929, 5950, 5951, 5952, 5953, 5954, 5955, 5956, 5963, 5967, 5969, 5970,5982, 5983, 5984, 5985, 5986, 5987, 5988, 5997, 5998, 5999, 6000, 6001,6002, 6068, 6069, 6070, 6071, 6072, 6074, 6075, 6076, 6077, 6078, 6080,6081, 6082, 6083, 6084, 6085, 6086, 6087, 6088, 6089, 6090, 6091, 6092,6093, 6094, 6095, 6096, 6097, 6098, 6100, 6101, 6102, 6103, 6105, 6106,6107, 6108, 6109, 6110, 6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118,6119, 6120, 6121, 6122, 6123, 6124, 6128, 6130, 6142, 6143, 6144, 6145,6146, 6147, 6148, 6149, 6151, 6152, 6153, 6154, 6155, 6156, 6157, 6169,6170, 6171, 6172, 6173, 6174, 6175, 6176, 6177, 6178, 6179, 6180, 6181,6182, 6185, 6186, 6187, 6191, 6208, 6209, 6231, 6232, 6233, 6234, 6235,6236, 6237, 6238, 6239, 6251, 6256, 6257, 6258, 6259, 6260, 6261, 6262,6278, 6279, 6280, 6281, 6282, 6286, 6287, 6288, 6289, 6290, 6298, 6299,6300, 6301, 6302, 6303, 6304, 6305, 6306, 6307, 6308, 6309, 6310, 6311,6312, 6313, 6314, 6315, 6356, 6357, 6358, 6378, 6379, 6387, 6388, 6389,6390, 6391, 6392, 6393, 6394, 6395, 6396, 6397, 6398, 6399, 6400, 6401,6402, 6403, 6405, 6406, 6407, 6451, 6452, 6453, 6497, 6498, 6499, 6500,6501, 6502, 6503, 6509, 6510, 6511, 6512, 6514, 6519, 6520, 6521, 6522,6523, 6549, 6550, 6551, 6552, 6553, 6554, 6555, 6556, 6557, 6558, 6588,6589, 6599, 6600, 6601, 6602, 6603, 6604, 6605, 6606, 6611, 6612, 6613,6614, 6615, 6616, 6617, 6618, 6619, 6620, 6621, 6622, 6623, 6624, 6625,6626, 6627, 6628, 6629, 6630, 6631, 6632, 6633, 6634, 6638, 6639, 6640,6641, 6642, 6643, 6644, 6684, 6685, 6686, 6697, 6698, 6699, 6700, 6701,6719, 6721, 6723, 6724, 6754, 6755, 6756, 6757, 6758, 6759, 6760, 6761,6762, 6763, 6764, 6796, 6797, 6798, 6799, 6800, 6805, 6806, 6807, 6808,6809, 6814, 6815, 6816, 6817, 6818, 6824, 6825, 6826, 6831, 6832, 6833,6834, 6853, 6854, 6855, 6856, 6857, 6860, 6861, 6862, 6863, 6864, 6865,6866, 6867, 6868, 6869, 6872, 6873, 6875, 6876, 6877, 6878, 6879, 6885,6888, 6889, 6890, 6891, 6892, 6893, 6894, 6895, 6896, 6897, 6898, 6899,6900, 6901, 6902, 6903, 6904, 6905, 6906, 6907, 6908, 6909, 6910, 6911,6912, 6913, 6914, 6915, 6916, 6917, 6918, 6919, 6920, 6921, 6922, 6923,6924, 6925, 6926, 6927, 6928, 6929, 6930, 6931, 6932, 6933, 6934, 6935,6936, 6953, 6954, 6955, 6957, 6958, 6959, 6960, 6962, 6963, 6964, 6965,6966, 6967, 6968, 6969, 6970, 6971, 6972, 6973, 6983, 6984, 6985, 6988,6989, 6990, 6991, 6992, 6993, 6994, 6995, 7007, 7008, 7009, 7010, 7011,7012, 7015, 7016, 7017, 7018, 7019, 7020, 7021, 7034, 7035, 7084, 7086,7087, 7088, 7089, 7090, 7091, 7092, 7128, 7137, 7138, 7139, 7140, 7141,7142, 7143, 7144, 7145, 7146, 7147, 7148, 7149, 7150, 7151, 7152, 7153,7154, 7155, 7195, 7196, 7197, 7198, 7200, 7201, 7202, 7203, 7204, 7205,7206, 7207, 7208, 7209, 7210, 7211, 7212, 7213, 7214, 7215, 7216, 7217,7218, 7219, 7220, 7221, 7222, 7223, 7224, 7225, 7226, 7227, 7228, 7229,7230, 7231, 7232, 7233, 7234, 7235, 7236, 7237, 7238, 7239, 7240, 7241,7243, 7244, 7245, 7246, 7247, 7248, 7249, 7250, 7251, 7252, 7253, 7254,7255, 7256, 7257, 7258, 7259, 7260, 7261, 7262, 7263, 7264, 7265, 7266,7267, 7268, 7269, 7270, 7271, 7291, 7292, 7293, 7294, 7295, 7296, 7297,7298, 7347, 7348, 7349, 7358, 7359, 7360, 7361, 7362, 7363, 7364, 7365,7372, 7373, 7374, 7375, 7376, 7377, 7378, 7383, 7415, 7416, 7417, 7438,7471, 7472, 7473, 7474, 7475, 7476, 7477, 7490, 7496, 7497, 7498, 7499,7500, 7501, 7502, 7503, 7504, 7505, 7506, 7507, 7508, 7509, 7510, 7511,7512, 7513, 7514, 7515, 7532, 7533, 7534, 7535, 7537, 7538, 7539, 7540,7541, 7542, 7543, 7544, 7545, 7546, 7547, 7548, 7549, 7550, 7551, 7552,7553, 7554, 7555, 7556, 7557, 7558, 7559, 7560, 7585, 7586, 7587, 7588,7589, 7590, 7591, 7592, 7593, 7595, 7596, 7597, 7599, 7600, 7601, 7617,7618, 7619, 7620, 7621, 7622, 7623, 7638, 7668, 7669, 7670, 7672, 7673,7674, 7675, 7676, 7685, 7687, 7694, 7695, 7696, 7717, 7718, 7719, 7720,7721, 7722, 7723, 7724, 7725, 7726, 7727, 7728, 7729, 7730, 7731, 7732,7733, 7734, 7735, 7736, 7737, 7738, 7739, 7740, 7741, 7742, 7743, 7744,7746, 7747, 7748, 7749, 7750, 7751, 7752, 7757, 7758, 7759, 7760, 7761,7762, 7763, 7764, 7765, 7766, 7767, 7768, 7796, 7797, 7805, 7813, 7814,7815, 7816, 7817, 7818, 7819, 7820, 7821, 7822, 7848, 7849, 7850, 7851,7852, 7853, 7854, 7859, 7860, 7861, 7862, 7863, 7864, 7865, 7866, 7867,7869, 7870, 7874, 7875, 7917, 7918, 7919, 7925, 7926, 7927, 7928, 7929,7941, 7942, 7943, 7944, 7945, 7946, 7947, 7948, 7949, 7950, 7951, 7952,7953, 7955, 7956, 7957, 7958, 7959, 7960, 7961, 7962, 7963, 7964, 7965,7966, 7967, 7968, 7969, 7970, 7971, 7972, 7974, 7975, 7976, 7977, 7978,7979, 7980, 7981, 7982, 7983, 7984, 7985, 7986, 7987, 7988, 7989, 7990,7991, 7992, 7993, 7994, 7995, 7996, 7997, 7998, 7999, 8000, 8011, 8012,8013, 8014, 8015, 8016, 8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024,8025, 8026, 8027, 8028, 8029, 8030, 8048, 8049, 8050, 8051, 8052, 8053,8054, 8055, 8056, 8057, 8087, 8088, 8089, 8090, 8091, 8092, 8095, 8096,8097, 8098, 8099, 8100, 8101, 8102, 8103, 8104, 8105, 8106, 8107, 8110,8111, 8112, 8113, 8114, 8115, 8116, 8117, 8118, 8119, 8120, 8121, 8122,8123, 8124, 8125, 8126, 8127, 8130, 8131, 8132, 8133, 8134, 8135, 8136,8144, 8145, 8166, 8169, 8170, 8171, 8172, 8173, 8174, 8175, 8176, 8177,8178, 8179, 8180, 8181, 8182, 8188, 8191, 8192, 8193, 8194, 8195, 8196,8197, 8198, 8199, 8200, 8201, 8209, 8210, 8211, 8212, 8215, 8227, 8230,8231, 8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241, 8242,8243, 8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251, 8260, 8261, 8262,8263, 8299, 8302, 8303, 8304, 8305, 8306, 8353, 8354, 8355, 8356, 8357,8358, 8359, 8360, 8361, 8375, 8376, 8377, 8378, 8379, 8380, 8381, 8382,8410, 8411, 8412, 8440, 8459, 8460, 8461, 8462, 8463, 8474, 8475, 8476,8477, 8478, 8508, 8509, 8510, 8511, 8512, 8514, 8515, 8546, 8547, 8548,8549, 8550, 8551, 8552, 8553, 8554, 8555, 8570, 8571, 8572, 8573, 8574,8575, 8576, 8577, 8578, 8579, 8580, 8581, 8586, 8587, 8593, 8594, 8595,8596, 8597, 8601, 8602, 8603, 8604, 8605, 8615, 8617, 8618, 8655, 8686,8709, 8712, 8713, 8714, 8715, 8716, 8717, 8718, 8719, 8720, 8721, 8722,8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730, 8731, 8732, 8733, 8734,8770, 8771, 8788, 8789, 8790, 8791, 8792, 8793, 8794, 8795, 8796, 8797,8798, 8799, 8800, 8801, 8802, 8803, 8804, 8805, 8806, 8810, 8811, 8817,8818, 8819, 8820, 8821, 8822, 8823, 8829, 8830, 8837, 8838, 8839, 8840,8841, 8848, 8849, 8850, 8851, 8854, 8855, 8856, 8857, 8858, 8859, 8860,8861, 8862, 8863, 8864, 8865, 8867, 8868, 8872, 8873, 8874, 8875, 8876,8878, 8879, 8880, 8881, 8882, 8883, 8884, 8885, 8886, 8887, 8888, 8889,8890, 8891, 8892, 8893, 8894, 8895, 8896, 8897, 8898, 8899, 8900, 8901,8902, 8903, 8904, 8906, 8913, 8914, 8915, 8916, 8917, 8918, 8919, 8920,8921, 8925, 8926, 8927, 8928, 8933, 8934, 8935, 8936, 8937, 8938, 8939,8940, 8941, 8942, 8943, 8944, 8945, 8946, 8947, 8948, 8949, 8950, 8951,8954, 8955, 8956, 8992, 8993, 8994, 8995, 8996, 8997, 8998, 8999, 9000,9001, 9002, 9003, 9010, 9011, 9012, 9013, 9014, 9018, 9019, 9020, 9021,9022, 9023, 9024, 9025, 9026, 9027, 9028, 9029, 9030, 9031, 9041, 9042,9043, 9044, 9045, 9046, 9047, 9048, 9049, 9050, 9051, 9052, 9053, 9054,9055, 9056, 9057, 9058, 9059, 9065, 9066, 9087, 9088, 9091, 9092, 9093,9094, 9095, 9096, 9097, 9098, 9103, 9104, 9105, 9106, 9107, 9108, 9109,9110, 9123, 9124, 9125, 9126, 9127, 9128, 9129, 9130, 9131, 9133, 9134,9135, 9136, 9137, 9145, 9146, 9147, 9148, 9149, 9150, 9151, 9160, 9161,9162, 9163, 9201, 9203.

Prostate Cancer

Prostate that forms in the tissue of the prostate mainly affects oldermen. Non-limiting examples of prostate cancer include, but are notlimited to, weak or interrupted flow of urine, frequent urination,trouble urinating, pain or burning during urination, blood in the urineor semen, pain in the back, hips or pelvis that does not go away andpainful ejaculation.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withprostate cancer by administering to said subject an isolatedpolynucleotide encoding an oncology-related polypeptide of interest. Inone embodiment, the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA of the present inventionmay be used to reduce, eliminate, or prevent tumor growth in a subjectwho has been diagnosed or may be diagnosed with prostate cancer byadministering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In one embodiment, theoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA of the present invention may be used to reduceand/or ameliorate at least one symptom of cancer in a subject who hasbeen diagnosed or may be diagnosed with prostate cancer by administeringto said subject an isolated polynucleotide encoding an oncology-relatedpolypeptide of interest. In a further embodiment, the oncology-relatedpolypeptide of interest may include, but not limited to, SEQ ID NOs:4704, 4705, 4706, 4707, 4708, 4709, 4710, 4711, 4712, 4713, 4714, 4715,4716, 4717, 4718, 4719, 4720, 4721, 4722, 4723, 4724, 4725, 4726, 4727,4728, 4729, 4730, 4731, 4732, 4733, 4734, 4735, 4736, 4737, 4738, 4739,4740, 4741, 4742, 4743, 4744, 4745, 4746, 4747, 4748, 4749, 4750, 4751,4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4825, 4826,4827, 4828, 4829, 4830, 4832, 4835, 4836, 4854, 4855, 4856, 4857, 4858,4859, 4860, 4861, 4863, 4864, 4865, 4866, 4867, 4868, 4869, 4870, 4871,4872, 4873, 4885, 4886, 4887, 4888, 4889, 4890, 4891, 4892, 4893, 4894,4895, 4896, 4897, 4898, 4899, 4900, 4901, 4902, 4903, 4904, 4905, 4906,4907, 4908, 4909, 4910, 4911, 4912, 4913, 4914, 4915, 4916, 4917, 4918,4919, 4920, 4960, 4961, 4962, 4963, 4964, 4965, 4966, 4967, 4968, 4969,4970, 4971, 4972, 4973, 4974, 4975, 4976, 4977, 4978, 4979, 4980, 4981,4982, 4983, 4984, 4985, 4986, 4987, 4988, 4989, 4990, 4991, 4992, 4993,4994, 4995, 4996, 4997, 4998, 4999, 5000, 5001, 5002, 5008, 5009, 5010,5011, 5012, 5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021, 5022,5023, 5024, 5025, 5026, 5027, 5028, 5030, 5031, 5032, 5033, 5034, 5035,5036, 5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046, 5047,5048, 5049, 5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058, 5059,5060, 5061, 5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071,5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079, 5103, 5104, 5105, 5106,5107, 5108, 5109, 5110, 5118, 5119, 5120, 5121, 5122, 5123, 5124, 5125,5126, 5127, 5128, 5129, 5130, 5131, 5132, 5133, 5134, 5135, 5136, 5137,5138, 5139, 5140, 5141, 5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152,5153, 5154, 5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164,5165, 5166, 5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176,5177, 5178, 5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191,5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203,5204, 5205, 5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5230, 5231,5233, 5234, 5235, 5236, 5249, 5250, 5251, 5252, 5253, 5254, 5255, 5256,5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264, 5265, 5266, 5267, 5268,5269, 5270, 5271, 5272, 5273, 5274, 5275, 5276, 5277, 5278, 5279, 5280,5281, 5291, 5292, 5293, 5294, 5295, 5296, 5297, 5298, 5299, 5300, 5301,5302, 5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311, 5312, 5313,5314, 5315, 5316, 5317, 5318, 5319, 5320, 5321, 5322, 5323, 5324, 5325,5326, 5327, 5328, 5329, 5330, 5331, 5332, 5333, 5334, 5335, 5336, 5337,5338, 5339, 5340, 5341, 5342, 5343, 5344, 5345, 5346, 5347, 5348, 5349,5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358, 5359, 5360, 5361,5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370, 5371, 5372, 5373,5374, 5375, 5376, 5377, 5378, 5379, 5380, 5381, 5382, 5383, 5384, 5385,5386, 5387, 5388, 5389, 5390, 5391, 5392, 5393, 5419, 5420, 5421, 5422,5423, 5424, 5426, 5427, 5428, 5429, 5438, 5447, 5448, 5449, 5450, 5451,5452, 5453, 5454, 5455, 5456, 5457, 5458, 5459, 5460, 5461, 5462, 5463,5464, 5465, 5469, 5470, 5477, 5478, 5479, 5480, 5481, 5482, 5490, 5491,5492, 5493, 5494, 5495, 5496, 5497, 5498, 5499, 5504, 5505, 5506, 5507,5508, 5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516, 5517, 5518, 5519,5520, 5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529, 5530, 5531,5532, 5533, 5534, 5535, 5536, 5537, 5538, 5539, 5540, 5541, 5542, 5543,5544, 5545, 5546, 5547, 5548, 5549, 5551, 5552, 5553, 5554, 5555, 5556,5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565, 5566, 5567, 5568,5569, 5570, 5571, 5577, 5578, 5579, 5580, 5590, 5591, 5592, 5593, 5594,5595, 5596, 5605, 5606, 5607, 5608, 5609, 5610, 5611, 5616, 5617, 5618,5619, 5620, 5621, 5622, 5623, 5631, 5632, 5633, 5638, 5639, 5640, 5671,5672, 5673, 5674, 5675, 5676, 5677, 5678, 5679, 5680, 5681, 5682, 5683,5684, 5685, 5686, 5687, 5688, 5689, 5690, 5691, 5692, 5693, 5694, 5695,5696, 5697, 5698, 5699, 5700, 5701, 5702, 5703, 5704, 5705, 5706, 5707,5708, 5709, 5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717, 5727, 5740,5741, 5742, 5743, 5744, 5745, 5746, 5747, 5748, 5749, 5750, 5751, 5755,5757, 5758, 5759, 5760, 5761, 5762, 5763, 5764, 5765, 5766, 5767, 5768,5769, 5770, 5772, 5773, 5775, 5776, 5777, 5778, 5779, 5780, 5783, 5784,5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796,5797, 5798, 5799, 5800, 5801, 5802, 5803, 5811, 5812, 5813, 5814, 5821,5822, 5823, 5824, 5825, 5826, 5827, 5828, 5829, 5830, 5831, 5833, 5834,5836, 5837, 5838, 5839, 5848, 5849, 5850, 5851, 5852, 5853, 5854, 5855,5856, 5857, 5858, 5859, 5860, 5861, 5862, 5863, 5864, 5865, 5866, 5867,5868, 5869, 5870, 5871, 5872, 5873, 5874, 5875, 5876, 5877, 5878, 5880,5881, 5882, 5883, 5884, 5885, 5886, 5887, 5888, 5889, 5890, 5891, 5892,5893, 5894, 5895, 5896, 5897, 5898, 5899, 5907, 5908, 5909, 5911, 5912,5913, 5914, 5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922, 5929, 5950,5951, 5952, 5953, 5954, 5955, 5956, 5963, 5967, 5969, 5970, 5974, 5975,5976, 5977, 5978, 5979, 5980, 5981, 5982, 5983, 5984, 5985, 5986, 5987,5988, 5994, 5995, 5996, 5997, 5998, 5999, 6000, 6001, 6002, 6020, 6021,6022, 6023, 6024, 6025, 6026, 6027, 6028, 6056, 6057, 6058, 6059, 6060,6061, 6062, 6063, 6064, 6065, 6066, 6067, 6068, 6069, 6070, 6071, 6072,6073, 6074, 6075, 6076, 6077, 6078, 6079, 6080, 6081, 6082, 6083, 6084,6085, 6086, 6087, 6088, 6089, 6090, 6091, 6092, 6093, 6094, 6095, 6096,6097, 6098, 6100, 6101, 6102, 6103, 6105, 6106, 6107, 6108, 6109, 6110,6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118, 6119, 6120, 6121, 6122,6123, 6124, 6128, 6130, 6142, 6143, 6144, 6145, 6146, 6147, 6148, 6149,6150, 6151, 6152, 6153, 6154, 6155, 6156, 6157, 6169, 6170, 6171, 6172,6173, 6174, 6175, 6176, 6177, 6178, 6179, 6180, 6181, 6182, 6185, 6186,6187, 6189, 6190, 6191, 6208, 6209, 6231, 6232, 6233, 6234, 6235, 6236,6237, 6238, 6239, 6251, 6256, 6257, 6258, 6259, 6260, 6261, 6262, 6278,6279, 6280, 6281, 6282, 6286, 6287, 6288, 6289, 6290, 6298, 6299, 6300,6301, 6302, 6303, 6304, 6305, 6306, 6307, 6308, 6309, 6310, 6311, 6312,6313, 6314, 6315, 6346, 6347, 6356, 6357, 6358, 6365, 6366, 6367, 6368,6369, 6370, 6372, 6378, 6379, 6387, 6388, 6389, 6390, 6391, 6392, 6393,6394, 6395, 6396, 6397, 6398, 6399, 6400, 6401, 6402, 6403, 6405, 6406,6407, 6446, 6447, 6448, 6451, 6452, 6453, 6497, 6498, 6499, 6500, 6501,6502, 6503, 6504, 6505, 6506, 6507, 6508, 6509, 6510, 6511, 6512, 6514,6519, 6520, 6521, 6522, 6523, 6549, 6550, 6551, 6552, 6553, 6554, 6555,6556, 6557, 6558, 6588, 6589, 6591, 6592, 6593, 6594, 6595, 6596, 6597,6598, 6599, 6600, 6601, 6602, 6603, 6604, 6605, 6606, 6611, 6612, 6613,6614, 6615, 6616, 6617, 6618, 6619, 6620, 6621, 6622, 6623, 6624, 6625,6626, 6627, 6628, 6629, 6630, 6631, 6632, 6633, 6634, 6638, 6639, 6640,6641, 6642, 6643, 6644, 6684, 6685, 6686, 6697, 6698, 6699, 6700, 6701,6716, 6719, 6721, 6723, 6724, 6725, 6726, 6727, 6728, 6729, 6730, 6731,6732, 6733, 6734, 6735, 6754, 6755, 6756, 6757, 6758, 6759, 6760, 6761,6762, 6763, 6764, 6796, 6797, 6798, 6799, 6800, 6801, 6805, 6806, 6807,6808, 6809, 6814, 6815, 6816, 6817, 6818, 6824, 6825, 6826, 6831, 6832,6833, 6834, 6853, 6854, 6855, 6856, 6857, 6858, 6859, 6860, 6861, 6862,6863, 6864, 6865, 6866, 6867, 6868, 6869, 6872, 6873, 6875, 6876, 6877,6878, 6879, 6885, 6888, 6889, 6890, 6891, 6892, 6893, 6894, 6895, 6896,6897, 6898, 6899, 6900, 6901, 6902, 6903, 6904, 6905, 6906, 6907, 6908,6909, 6910, 6911, 6912, 6913, 6914, 6915, 6916, 6917, 6918, 6919, 6920,6921, 6922, 6923, 6924, 6925, 6926, 6927, 6928, 6929, 6930, 6931, 6932,6933, 6934, 6935, 6936, 6953, 6954, 6955, 6957, 6958, 6959, 6960, 6962,6963, 6964, 6965, 6966, 6967, 6968, 6969, 6970, 6971, 6972, 6973, 6983,6984, 6985, 6986, 6987, 6988, 6989, 6990, 6991, 6992, 6993, 6994, 6995,7007, 7008, 7009, 7010, 7011, 7012, 7015, 7016, 7017, 7018, 7019, 7020,7021, 7022, 7023, 7024, 7025, 7026, 7034, 7035, 7084, 7086, 7087, 7088,7089, 7090, 7091, 7092, 7128, 7137, 7138, 7139, 7140, 7141, 7142, 7143,7144, 7145, 7146, 7147, 7148, 7149, 7150, 7151, 7152, 7153, 7154, 7155,7183, 7184, 7185, 7186, 7188, 7189, 7195, 7196, 7197, 7198, 7200, 7201,7202, 7203, 7204, 7205, 7206, 7207, 7208, 7209, 7210, 7211, 7212, 7213,7214, 7215, 7216, 7217, 7218, 7219, 7220, 7221, 7222, 7223, 7224, 7225,7226, 7227, 7228, 7229, 7230, 7231, 7232, 7233, 7234, 7235, 7236, 7237,7238, 7239, 7240, 7241, 7243, 7244, 7245, 7246, 7247, 7248, 7249, 7250,7251, 7252, 7253, 7254, 7255, 7256, 7257, 7258, 7259, 7260, 7261, 7262,7263, 7264, 7265, 7266, 7267, 7268, 7269, 7270, 7271, 7272, 7273, 7274,7275, 7291, 7292, 7293, 7294, 7295, 7296, 7297, 7298, 7347, 7348, 7349,7355, 7356, 7357, 7358, 7359, 7360, 7361, 7362, 7363, 7364, 7365, 7372,7373, 7374, 7375, 7376, 7377, 7378, 7383, 7415, 7416, 7417, 7438, 7439,7440, 7441, 7442, 7471, 7472, 7473, 7474, 7475, 7476, 7477, 7478, 7490,7496, 7497, 7498, 7499, 7500, 7501, 7502, 7503, 7504, 7505, 7506, 7507,7508, 7509, 7510, 7511, 7512, 7513, 7514, 7515, 7532, 7533, 7534, 7535,7537, 7538, 7539, 7540, 7541, 7542, 7543, 7544, 7545, 7546, 7547, 7548,7549, 7550, 7551, 7552, 7553, 7554, 7555, 7556, 7557, 7558, 7559, 7560,7561, 7562, 7563, 7564, 7565, 7566, 7567, 7568, 7569, 7570, 7581, 7582,7583, 7584, 7585, 7586, 7587, 7588, 7589, 7590, 7591, 7592, 7593, 7595,7596, 7597, 7599, 7600, 7601, 7617, 7618, 7619, 7620, 7621, 7622, 7623,7638, 7668, 7669, 7670, 7672, 7673, 7674, 7675, 7676, 7685, 7687, 7694,7695, 7696, 7717, 7718, 7719, 7720, 7721, 7722, 7723, 7724, 7725, 7726,7727, 7728, 7729, 7730, 7731, 7732, 7733, 7734, 7735, 7736, 7737, 7738,7739, 7740, 7741, 7742, 7743, 7744, 7746, 7747, 7748, 7749, 7750, 7751,7752, 7757, 7758, 7759, 7760, 7761, 7762, 7763, 7764, 7765, 7766, 7767,7768, 7796, 7797, 7805, 7813, 7814, 7815, 7816, 7817, 7818, 7819, 7820,7821, 7822, 7848, 7849, 7850, 7851, 7852, 7853, 7854, 7859, 7860, 7861,7862, 7863, 7864, 7865, 7866, 7867, 7869, 7870, 7871, 7872, 7873, 7874,7875, 7876, 7877, 7917, 7918, 7919, 7925, 7926, 7927, 7928, 7929, 7941,7942, 7943, 7944, 7945, 7946, 7947, 7948, 7949, 7950, 7951, 7952, 7953,7955, 7956, 7957, 7958, 7959, 7960, 7961, 7962, 7963, 7964, 7965, 7966,7967, 7968, 7969, 7970, 7971, 7972, 7974, 7975, 7976, 7977, 7978, 7979,7980, 7981, 7982, 7983, 7984, 7985, 7986, 7987, 7988, 7989, 7990, 7991,7992, 7993, 7994, 7995, 7996, 7997, 7998, 7999, 8000, 8011, 8012, 8013,8014, 8015, 8016, 8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024, 8025,8026, 8027, 8028, 8029, 8030, 8048, 8049, 8050, 8051, 8052, 8053, 8054,8055, 8056, 8057, 8087, 8088, 8089, 8090, 8091, 8092, 8095, 8096, 8097,8098, 8099, 8100, 8101, 8102, 8103, 8104, 8105, 8106, 8107, 8110, 8111,8112, 8113, 8114, 8115, 8116, 8117, 8118, 8119, 8120, 8121, 8122, 8123,8124, 8125, 8126, 8127, 8130, 8131, 8132, 8133, 8134, 8135, 8136, 8144,8145, 8166, 8169, 8170, 8171, 8172, 8173, 8174, 8175, 8176, 8177, 8178,8179, 8180, 8181, 8182, 8188, 8191, 8192, 8193, 8194, 8195, 8196, 8197,8198, 8199, 8200, 8201, 8209, 8210, 8211, 8212, 8215, 8227, 8230, 8231,8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241, 8242, 8243,8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251, 8252, 8253, 8254, 8255,8256, 8257, 8258, 8259, 8260, 8261, 8262, 8263, 8299, 8302, 8303, 8304,8305, 8306, 8334, 8335, 8336, 8337, 8338, 8339, 8340, 8341, 8342, 8343,8344, 8353, 8354, 8355, 8356, 8357, 8358, 8359, 8360, 8361, 8365, 8366,8367, 8368, 8369, 8370, 8371, 8375, 8376, 8377, 8378, 8379, 8380, 8381,8382, 8410, 8411, 8412, 8440, 8459, 8460, 8461, 8462, 8463, 8474, 8475,8476, 8477, 8478, 8501, 8502, 8503, 8507, 8508, 8509, 8510, 8511, 8512,8514, 8515, 8537, 8538, 8539, 8544, 8545, 8546, 8547, 8548, 8549, 8550,8551, 8552, 8553, 8554, 8555, 8558, 8559, 8560, 8561, 8562, 8563, 8564,8565, 8566, 8567, 8570, 8571, 8572, 8573, 8574, 8575, 8576, 8577, 8578,8579, 8580, 8581, 8586, 8587, 8588, 8589, 8590, 8591, 8592, 8593, 8594,8595, 8596, 8597, 8601, 8602, 8603, 8604, 8605, 8615, 8617, 8618, 8655,8686, 8693, 8694, 8695, 8696, 8697, 8698, 8709, 8712, 8713, 8714, 8715,8716, 8717, 8718, 8719, 8720, 8721, 8722, 8723, 8724, 8725, 8726, 8727,8728, 8729, 8730, 8731, 8732, 8733, 8734, 8770, 8771, 8788, 8789, 8790,8791, 8792, 8793, 8794, 8795, 8796, 8797, 8798, 8799, 8800, 8801, 8802,8803, 8804, 8805, 8806, 8807, 8808, 8809, 8810, 8811, 8817, 8818, 8819,8820, 8821, 8822, 8823, 8824, 8825, 8826, 8827, 8828, 8829, 8830, 8837,8838, 8839, 8840, 8841, 8842, 8843, 8844, 8845, 8846, 8847, 8848, 8849,8850, 8851, 8854, 8855, 8856, 8857, 8858, 8859, 8860, 8861, 8862, 8863,8864, 8865, 8867, 8868, 8872, 8873, 8874, 8875, 8876, 8877, 8878, 8879,8880, 8881, 8882, 8883, 8884, 8885, 8886, 8887, 8888, 8889, 8890, 8891,8892, 8893, 8894, 8895, 8896, 8897, 8898, 8899, 8900, 8901, 8902, 8903,8904, 8906, 8913, 8914, 8915, 8916, 8917, 8918, 8919, 8920, 8921, 8923,8924, 8925, 8926, 8927, 8928, 8933, 8934, 8935, 8936, 8937, 8938, 8939,8940, 8941, 8942, 8943, 8944, 8945, 8946, 8947, 8948, 8949, 8950, 8951,8954, 8955, 8956, 8974, 8975, 8976, 8977, 8978, 8979, 8980, 8981, 8992,8993, 8994, 8995, 8996, 8997, 8998, 8999, 9000, 9001, 9002, 9003, 9010,9011, 9012, 9013, 9014, 9018, 9019, 9020, 9021, 9022, 9023, 9024, 9025,9026, 9027, 9028, 9029, 9030, 9031, 9041, 9042, 9043, 9044, 9045, 9046,9047, 9048, 9049, 9050, 9051, 9052, 9053, 9054, 9055, 9056, 9057, 9058,9059, 9065, 9066, 9087, 9088, 9091, 9092, 9093, 9094, 9095, 9096, 9097,9098, 9103, 9104, 9105, 9106, 9107, 9108, 9109, 9110, 9111, 9112, 9113,9114, 9115, 9123, 9124, 9125, 9126, 9127, 9128, 9129, 9130, 9131, 9133,9134, 9135, 9136, 9137, 9145, 9146, 9147, 9148, 9149, 9150, 9151, 9160,9161, 9162, 9163, 9186, 9187, 9188, 9189, 9190, 9191, 9192, 9193, 9194,9195, 9196, 9197, 9198, 9199, 9200, 9201, 9203.

Testicular Cancer

Testicular cancer forms in the tissues of one or both testicles and ismost common in young or middle-aged men. Most testicular cancers beingin germ cells and are called testicular germ cell tumors. There are twotypes of testicular germ cell tumors called seminomas and nonseminomas.Common symptoms of testicular cancer include, but are not limited to, apainless lump or swelling in either testicle, change in how the testiclefeels, dull ache in the lower abdomen or the groin, sudden build-up offluid in the scrotum and pain or discomfort in a testicle or in thescrotum.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withtesticular cancer by administering to said subject an isolatedpolynucleotide encoding an oncology-related polypeptide of interest. Inone embodiment, the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA of the present inventionmay be used to reduce, eliminate, or prevent tumor growth in a subjectwho has been diagnosed or may be diagnosed with testicular cancer byadministering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In one embodiment, theoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA of the present invention may be used to reduceand/or ameliorate at least one symptom of cancer in a subject who hasbeen diagnosed or may be diagnosed with testicular cancer byadministering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In a further embodiment, theoncology-related polypeptide of interest may include, but not limitedto, SEQ ID NOs: 4711, 4712, 4713, 4714, 4715, 4716, 4717, 4718, 4719,4720, 4721, 4722, 4723, 4724, 4725, 4726, 4727, 4728, 4742, 4743, 4744,4745, 4746, 4747, 4748, 4749, 4750, 4751, 4781, 4782, 4783, 4784, 4785,4786, 4787, 4788, 4789, 4790, 4825, 4826, 4827, 4828, 4829, 4830, 4832,4854, 4855, 4856, 4857, 4858, 4859, 4860, 4861, 4863, 4864, 4865, 4866,4867, 4868, 4869, 4870, 4871, 4872, 4873, 4885, 4886, 4887, 4888, 4889,4890, 4891, 4892, 4893, 4894, 4911, 4912, 4913, 4914, 4915, 4916, 4917,4918, 4919, 4920, 4960, 4961, 4962, 4963, 4964, 4965, 4966, 4967, 4968,4969, 4970, 4971, 4972, 4973, 4974, 4975, 4976, 4977, 4978, 4979, 4980,4981, 4982, 4983, 4984, 4985, 4986, 4987, 4988, 4989, 4990, 4991, 4992,4993, 4994, 4995, 4996, 4997, 4998, 4999, 5000, 5001, 5002, 5008, 5009,5010, 5011, 5012, 5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021,5022, 5023, 5024, 5025, 5026, 5027, 5028, 5030, 5031, 5032, 5033, 5034,5035, 5036, 5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046,5047, 5048, 5049, 5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058,5059, 5060, 5061, 5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070,5071, 5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079, 5103, 5104, 5105,5106, 5107, 5108, 5109, 5110, 5118, 5119, 5120, 5121, 5122, 5123, 5124,5125, 5126, 5127, 5128, 5129, 5130, 5131, 5132, 5133, 5134, 5135, 5136,5137, 5138, 5139, 5140, 5141, 5145, 5146, 5147, 5148, 5149, 5150, 5151,5152, 5153, 5154, 5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163,5164, 5165, 5166, 5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175,5176, 5177, 5178, 5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190,5191, 5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202,5203, 5204, 5205, 5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5230,5231, 5252, 5253, 5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262,5263, 5264, 5265, 5266, 5267, 5268, 5269, 5270, 5271, 5291, 5292, 5293,5294, 5295, 5296, 5297, 5298, 5299, 5300, 5301, 5302, 5303, 5304, 5305,5306, 5307, 5308, 5309, 5310, 5311, 5312, 5313, 5314, 5315, 5316, 5317,5318, 5319, 5320, 5321, 5322, 5323, 5324, 5325, 5326, 5327, 5328, 5329,5330, 5331, 5332, 5333, 5334, 5335, 5336, 5337, 5338, 5339, 5340, 5341,5342, 5343, 5344, 5345, 5346, 5347, 5348, 5349, 5350, 5351, 5352, 5353,5354, 5355, 5356, 5357, 5358, 5359, 5360, 5361, 5362, 5363, 5364, 5365,5366, 5367, 5368, 5369, 5370, 5371, 5372, 5373, 5374, 5375, 5376, 5377,5378, 5379, 5380, 5381, 5382, 5383, 5384, 5385, 5386, 5387, 5388, 5389,5390, 5391, 5392, 5393, 5394, 5395, 5396, 5397, 5398, 5399, 5400, 5401,5402, 5419, 5420, 5421, 5426, 5427, 5428, 5429, 5438, 5447, 5448, 5449,5450, 5451, 5452, 5453, 5454, 5455, 5456, 5457, 5458, 5459, 5460, 5461,5462, 5463, 5464, 5465, 5469, 5470, 5477, 5478, 5479, 5480, 5481, 5482,5490, 5491, 5492, 5493, 5494, 5495, 5496, 5497, 5498, 5499, 5504, 5505,5506, 5507, 5508, 5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516, 5517,5518, 5519, 5520, 5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529,5530, 5531, 5532, 5533, 5534, 5535, 5536, 5548, 5549, 5551, 5552, 5553,5554, 5555, 5556, 5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565,5566, 5567, 5568, 5569, 5570, 5571, 5577, 5578, 5579, 5580, 5590, 5591,5592, 5593, 5594, 5595, 5596, 5605, 5606, 5607, 5608, 5609, 5610, 5611,5616, 5617, 5618, 5619, 5620, 5621, 5622, 5623, 5631, 5632, 5633, 5638,5639, 5640, 5671, 5672, 5673, 5674, 5675, 5676, 5677, 5678, 5679, 5680,5681, 5688, 5689, 5690, 5691, 5692, 5693, 5694, 5695, 5696, 5697, 5698,5699, 5700, 5701, 5702, 5703, 5708, 5709, 5710, 5711, 5712, 5713, 5714,5715, 5716, 5717, 5727, 5740, 5741, 5743, 5744, 5745, 5746, 5747, 5748,5749, 5750, 5751, 5755, 5757, 5758, 5759, 5760, 5761, 5762, 5763, 5764,5765, 5766, 5767, 5768, 5769, 5770, 5772, 5773, 5775, 5776, 5777, 5778,5779, 5780, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792,5793, 5794, 5795, 5796, 5797, 5798, 5799, 5800, 5801, 5802, 5803, 5811,5812, 5813, 5814, 5821, 5822, 5823, 5824, 5825, 5826, 5827, 5828, 5829,5830, 5831, 5833, 5834, 5836, 5837, 5838, 5839, 5848, 5849, 5850, 5851,5852, 5853, 5854, 5855, 5864, 5865, 5866, 5867, 5868, 5875, 5876, 5877,5878, 5880, 5881, 5882, 5883, 5884, 5885, 5886, 5887, 5888, 5889, 5890,5891, 5892, 5893, 5894, 5895, 5896, 5897, 5898, 5899, 5907, 5908, 5909,5911, 5912, 5913, 5914, 5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922,5929, 5950, 5951, 5952, 5953, 5954, 5955, 5956, 5963, 5967, 5969, 5970,5982, 5983, 5984, 5985, 5986, 5987, 5988, 5997, 5998, 5999, 6000, 6001,6002, 6068, 6069, 6070, 6071, 6072, 6074, 6075, 6076, 6077, 6078, 6080,6081, 6082, 6083, 6084, 6085, 6086, 6087, 6088, 6089, 6090, 6091, 6092,6093, 6094, 6095, 6096, 6097, 6098, 6100, 6101, 6102, 6103, 6105, 6106,6107, 6108, 6109, 6110, 6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118,6119, 6120, 6121, 6122, 6123, 6124, 6128, 6130, 6142, 6143, 6144, 6145,6146, 6147, 6148, 6149, 6151, 6152, 6153, 6154, 6155, 6156, 6157, 6169,6170, 6171, 6172, 6173, 6174, 6175, 6176, 6177, 6178, 6179, 6180, 6181,6182, 6185, 6186, 6187, 6191, 6208, 6209, 6231, 6232, 6233, 6234, 6235,6236, 6237, 6238, 6239, 6251, 6256, 6257, 6258, 6259, 6260, 6261, 6262,6278, 6279, 6280, 6281, 6282, 6286, 6287, 6288, 6289, 6290, 6298, 6299,6300, 6301, 6302, 6303, 6304, 6305, 6306, 6307, 6308, 6309, 6310, 6311,6312, 6313, 6314, 6315, 6356, 6357, 6358, 6378, 6379, 6387, 6388, 6389,6390, 6391, 6392, 6393, 6394, 6395, 6396, 6397, 6398, 6399, 6400, 6401,6402, 6403, 6405, 6406, 6407, 6451, 6452, 6453, 6497, 6498, 6499, 6500,6501, 6502, 6503, 6509, 6510, 6511, 6512, 6514, 6519, 6520, 6521, 6522,6523, 6549, 6550, 6551, 6552, 6553, 6554, 6555, 6556, 6557, 6558, 6588,6589, 6599, 6600, 6601, 6602, 6603, 6604, 6605, 6606, 6611, 6612, 6613,6614, 6615, 6616, 6617, 6618, 6619, 6620, 6621, 6622, 6623, 6624, 6625,6626, 6627, 6628, 6629, 6630, 6631, 6632, 6633, 6634, 6638, 6639, 6640,6641, 6642, 6643, 6644, 6658, 6684, 6685, 6686, 6697, 6698, 6699, 6700,6701, 6702, 6703, 6704, 6705, 6706, 6707, 6708, 6709, 6710, 6719, 6721,6723, 6724, 6754, 6755, 6756, 6757, 6758, 6759, 6760, 6761, 6762, 6763,6764, 6796, 6797, 6798, 6799, 6800, 6805, 6806, 6807, 6808, 6809, 6814,6815, 6816, 6817, 6818, 6824, 6825, 6826, 6831, 6832, 6833, 6834, 6853,6854, 6855, 6856, 6857, 6860, 6861, 6862, 6863, 6864, 6865, 6866, 6867,6868, 6869, 6872, 6873, 6875, 6876, 6877, 6878, 6879, 6885, 6888, 6889,6890, 6891, 6892, 6893, 6894, 6895, 6896, 6897, 6898, 6899, 6900, 6901,6902, 6903, 6904, 6905, 6906, 6907, 6908, 6909, 6910, 6911, 6912, 6913,6914, 6915, 6916, 6917, 6918, 6919, 6920, 6921, 6922, 6923, 6924, 6925,6926, 6927, 6928, 6929, 6930, 6931, 6932, 6933, 6934, 6935, 6936, 6953,6954, 6955, 6957, 6958, 6959, 6960, 6962, 6963, 6964, 6965, 6966, 6967,6968, 6969, 6970, 6971, 6972, 6973, 6983, 6984, 6985, 6988, 6989, 6990,6991, 6992, 6993, 6994, 6995, 7007, 7008, 7009, 7010, 7011, 7012, 7015,7016, 7017, 7018, 7019, 7020, 7021, 7034, 7035, 7084, 7086, 7087, 7088,7089, 7090, 7091, 7092, 7128, 7137, 7138, 7139, 7140, 7141, 7142, 7143,7144, 7145, 7146, 7147, 7148, 7149, 7150, 7151, 7152, 7153, 7154, 7155,7195, 7196, 7197, 7198, 7200, 7201, 7202, 7203, 7204, 7205, 7206, 7207,7208, 7209, 7210, 7211, 7212, 7213, 7214, 7215, 7216, 7217, 7218, 7219,7220, 7221, 7222, 7223, 7224, 7225, 7226, 7227, 7228, 7229, 7230, 7231,7232, 7233, 7234, 7235, 7236, 7237, 7238, 7239, 7240, 7241, 7243, 7244,7245, 7246, 7247, 7248, 7249, 7250, 7251, 7252, 7253, 7254, 7255, 7256,7257, 7258, 7259, 7260, 7261, 7262, 7263, 7264, 7265, 7266, 7267, 7268,7269, 7270, 7271, 7291, 7292, 7293, 7294, 7295, 7296, 7297, 7298, 7305,7306, 7307, 7308, 7309, 7310, 7311, 7312, 7313, 7314, 7315, 7316, 7317,7347, 7348, 7349, 7358, 7359, 7360, 7361, 7362, 7363, 7364, 7365, 7372,7373, 7374, 7375, 7376, 7377, 7378, 7383, 7415, 7416, 7417, 7438, 7471,7472, 7473, 7474, 7475, 7476, 7477, 7490, 7496, 7497, 7498, 7499, 7500,7501, 7502, 7503, 7504, 7505, 7506, 7507, 7508, 7509, 7510, 7511, 7512,7513, 7514, 7515, 7532, 7533, 7534, 7535, 7537, 7538, 7539, 7540, 7541,7542, 7543, 7544, 7545, 7546, 7547, 7548, 7549, 7550, 7551, 7552, 7553,7554, 7555, 7556, 7557, 7558, 7559, 7560, 7585, 7586, 7587, 7588, 7589,7590, 7591, 7592, 7593, 7594, 7595, 7596, 7597, 7599, 7600, 7601, 7617,7618, 7619, 7620, 7621, 7622, 7623, 7638, 7668, 7669, 7670, 7672, 7673,7674, 7675, 7676, 7685, 7687, 7694, 7695, 7696, 7717, 7718, 7719, 7720,7721, 7722, 7723, 7724, 7725, 7726, 7727, 7728, 7729, 7730, 7731, 7732,7733, 7734, 7735, 7736, 7737, 7738, 7739, 7740, 7741, 7742, 7743, 7744,7746, 7747, 7748, 7749, 7750, 7751, 7752, 7757, 7758, 7759, 7760, 7761,7762, 7763, 7764, 7765, 7766, 7767, 7768, 7796, 7797, 7805, 7813, 7814,7815, 7816, 7817, 7818, 7819, 7820, 7821, 7822, 7848, 7849, 7850, 7851,7852, 7853, 7854, 7859, 7860, 7861, 7862, 7863, 7864, 7865, 7866, 7867,7869, 7870, 7874, 7875, 7917, 7918, 7919, 7925, 7926, 7927, 7928, 7929,7941, 7942, 7943, 7944, 7945, 7946, 7947, 7948, 7949, 7950, 7951, 7952,7953, 7955, 7956, 7957, 7958, 7959, 7960, 7961, 7962, 7963, 7964, 7965,7966, 7967, 7968, 7969, 7970, 7971, 7972, 7974, 7975, 7976, 7977, 7978,7979, 7980, 7981, 7982, 7983, 7984, 7985, 7986, 7987, 7988, 7989, 7990,7991, 7992, 7993, 7994, 7995, 7996, 7997, 7998, 7999, 8000, 8011, 8012,8013, 8014, 8015, 8016, 8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024,8025, 8026, 8027, 8028, 8029, 8030, 8048, 8049, 8050, 8051, 8052, 8053,8054, 8055, 8056, 8057, 8087, 8088, 8089, 8090, 8091, 8092, 8095, 8096,8097, 8098, 8099, 8100, 8101, 8102, 8103, 8104, 8105, 8106, 8107, 8110,8111, 8112, 8113, 8114, 8115, 8116, 8117, 8118, 8119, 8120, 8121, 8122,8123, 8124, 8125, 8126, 8127, 8130, 8131, 8132, 8133, 8134, 8135, 8136,8144, 8145, 8166, 8169, 8170, 8171, 8172, 8173, 8174, 8175, 8176, 8177,8178, 8179, 8180, 8181, 8182, 8188, 8191, 8192, 8193, 8194, 8195, 8196,8197, 8198, 8199, 8200, 8201, 8209, 8210, 8211, 8212, 8215, 8227, 8230,8231, 8232, 8233, 8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241, 8242,8243, 8244, 8245, 8246, 8247, 8248, 8249, 8250, 8251, 8260, 8261, 8262,8263, 8299, 8302, 8303, 8304, 8305, 8306, 8353, 8354, 8355, 8356, 8357,8358, 8359, 8360, 8361, 8375, 8376, 8377, 8378, 8379, 8380, 8381, 8382,8410, 8411, 8412, 8440, 8459, 8460, 8461, 8462, 8463, 8474, 8475, 8476,8477, 8478, 8508, 8509, 8510, 8511, 8512, 8514, 8515, 8546, 8547, 8548,8549, 8550, 8551, 8552, 8553, 8554, 8555, 8570, 8571, 8572, 8573, 8574,8575, 8576, 8577, 8578, 8579, 8580, 8581, 8586, 8587, 8593, 8594, 8595,8596, 8597, 8601, 8602, 8603, 8604, 8605, 8615, 8617, 8618, 8655, 8686,8709, 8712, 8713, 8714, 8715, 8716, 8717, 8718, 8719, 8720, 8721, 8722,8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730, 8731, 8732, 8733, 8734,8770, 8771, 8788, 8789, 8790, 8791, 8792, 8793, 8794, 8795, 8796, 8797,8798, 8799, 8800, 8801, 8802, 8803, 8804, 8805, 8806, 8810, 8811, 8817,8818, 8819, 8820, 8821, 8822, 8823, 8829, 8830, 8837, 8838, 8839, 8840,8841, 8848, 8849, 8850, 8851, 8854, 8855, 8856, 8857, 8858, 8859, 8860,8861, 8862, 8863, 8864, 8865, 8867, 8868, 8872, 8873, 8874, 8875, 8876,8878, 8879, 8880, 8881, 8882, 8883, 8884, 8885, 8886, 8887, 8888, 8889,8890, 8891, 8892, 8893, 8894, 8895, 8896, 8897, 8898, 8899, 8900, 8901,8902, 8903, 8904, 8906, 8913, 8914, 8915, 8916, 8917, 8918, 8919, 8920,8921, 8925, 8926, 8927, 8928, 8933, 8934, 8935, 8936, 8937, 8938, 8939,8940, 8941, 8942, 8943, 8944, 8945, 8946, 8947, 8948, 8949, 8950, 8951,8954, 8955, 8956, 8992, 8993, 8994, 8995, 8996, 8997, 8998, 8999, 9000,9001, 9002, 9003, 9010, 9011, 9012, 9013, 9014, 9018, 9019, 9020, 9021,9022, 9023, 9024, 9025, 9026, 9027, 9028, 9029, 9030, 9031, 9041, 9042,9043, 9044, 9045, 9046, 9047, 9048, 9049, 9050, 9051, 9052, 9053, 9054,9055, 9056, 9057, 9058, 9059, 9065, 9066, 9087, 9088, 9091, 9092, 9093,9094, 9095, 9096, 9097, 9098, 9103, 9104, 9105, 9106, 9107, 9108, 9109,9110, 9123, 9124, 9125, 9126, 9127, 9128, 9129, 9130, 9131, 9133, 9134,9135, 9136, 9137, 9145, 9146, 9147, 9148, 9149, 9150, 9151, 9160, 9161,9162, 9163, 9201, 9203.

Throat Cancer

Throat cancer forms in the tissues of the pharynx and includes cancer ofthe nasopharynx (nasopharyngeal cancer), oropharynx (oropharyngealcancer), hypopharynx (hypopharyngeal cancer), and larynx (laryngealcancer). Common symptoms of throat cancer include, but are not limitedto, a sore throat that does not go away, ear pain, lump in the neck,painful or difficulty swallowing, change or hoarseness in the voice,trouble breathing or speaking, nosebleeds, trouble hearing, pain orringing in the ear, headaches, dull pain behind the breast bone, coughand weight loss for no reason.

In one embodiment, the oncology-related polynucleotides,oncology-related primary constructs or oncology-related mmRNA of thepresent invention may be used to treat a disease, disorder and/orcondition in a subject who has been diagnosed or may be diagnosed withthroat cancer by administering to said subject an isolatedpolynucleotide encoding an oncology-related polypeptide of interest. Inone embodiment, the oncology-related polynucleotides, oncology-relatedprimary constructs or oncology-related mmRNA of the present inventionmay be used to reduce, eliminate, or prevent tumor growth in a subjectwho has been diagnosed or may be diagnosed with throat cancer byadministering to said subject an isolated polynucleotide encoding anoncology-related polypeptide of interest. In one embodiment, theoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA of the present invention may be used to reduceand/or ameliorate at least one symptom of cancer in a subject who hasbeen diagnosed or may be diagnosed with throat cancer by administeringto said subject an isolated polynucleotide encoding an oncology-relatedpolypeptide of interest. In a further embodiment, the oncology-relatedpolypeptide of interest may include, but not limited to, SEQ ID NOs:4711, 4712, 4713, 4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722,4723, 4724, 4725, 4726, 4727, 4728, 4742, 4743, 4744, 4745, 4746, 4747,4748, 4749, 4750, 4751, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788,4789, 4790, 4825, 4826, 4827, 4828, 4829, 4830, 4832, 4854, 4855, 4856,4857, 4858, 4859, 4860, 4861, 4863, 4864, 4865, 4866, 4867, 4868, 4869,4870, 4871, 4872, 4873, 4885, 4886, 4887, 4888, 4889, 4890, 4891, 4892,4893, 4894, 4911, 4912, 4913, 4914, 4915, 4916, 4917, 4918, 4919, 4920,4960, 4961, 4962, 4963, 4964, 4965, 4966, 4967, 4968, 4969, 4970, 4971,4972, 4973, 4974, 4975, 4976, 4977, 4978, 4979, 4980, 4981, 4982, 4983,4984, 4985, 4986, 4987, 4988, 4989, 4990, 4991, 4992, 4993, 4994, 4995,4996, 4997, 4998, 4999, 5000, 5001, 5002, 5008, 5009, 5010, 5011, 5012,5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021, 5022, 5023, 5024,5025, 5026, 5027, 5028, 5030, 5031, 5032, 5033, 5034, 5035, 5036, 5037,5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046, 5047, 5048, 5049,5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058, 5059, 5060, 5061,5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070, 5071, 5072, 5073,5074, 5075, 5076, 5077, 5078, 5079, 5103, 5104, 5105, 5106, 5107, 5108,5109, 5110, 5118, 5119, 5120, 5121, 5122, 5123, 5124, 5125, 5126, 5127,5128, 5129, 5130, 5131, 5132, 5133, 5134, 5135, 5136, 5137, 5138, 5139,5140, 5141, 5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152, 5153, 5154,5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164, 5165, 5166,5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176, 5177, 5178,5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191, 5192, 5193,5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203, 5204, 5205,5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5230, 5231, 5252, 5253,5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262, 5263, 5264, 5265,5266, 5267, 5268, 5269, 5270, 5271, 5291, 5292, 5293, 5294, 5295, 5296,5297, 5298, 5299, 5300, 5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308,5309, 5310, 5311, 5312, 5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320,5321, 5322, 5323, 5324, 5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332,5333, 5334, 5335, 5336, 5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344,5345, 5346, 5347, 5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356,5357, 5358, 5359, 5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368,5369, 5370, 5371, 5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380,5381, 5382, 5383, 5384, 5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392,5393, 5394, 5395, 5396, 5397, 5398, 5399, 5400, 5401, 5402, 5419, 5420,5421, 5426, 5427, 5428, 5429, 5438, 5447, 5448, 5449, 5450, 5451, 5452,5453, 5454, 5455, 5456, 5457, 5458, 5459, 5460, 5461, 5462, 5463, 5464,5465, 5469, 5470, 5477, 5478, 5479, 5480, 5481, 5482, 5490, 5491, 5492,5493, 5494, 5495, 5496, 5497, 5498, 5499, 5504, 5505, 5506, 5507, 5508,5509, 5510, 5511, 5512, 5513, 5514, 5515, 5516, 5517, 5518, 5519, 5520,5521, 5522, 5523, 5524, 5525, 5526, 5527, 5528, 5529, 5530, 5531, 5532,5533, 5534, 5535, 5536, 5548, 5549, 5551, 5552, 5553, 5554, 5555, 5556,5557, 5558, 5559, 5560, 5561, 5562, 5563, 5564, 5565, 5566, 5567, 5568,5569, 5570, 5571, 5577, 5578, 5579, 5580, 5590, 5591, 5592, 5593, 5594,5595, 5596, 5605, 5606, 5607, 5608, 5609, 5610, 5611, 5616, 5617, 5618,5619, 5620, 5621, 5622, 5623, 5631, 5632, 5633, 5638, 5639, 5640, 5671,5672, 5673, 5674, 5675, 5676, 5677, 5678, 5679, 5680, 5681, 5688, 5689,5690, 5691, 5692, 5693, 5694, 5695, 5696, 5697, 5698, 5699, 5700, 5701,5702, 5703, 5708, 5709, 5710, 5711, 5712, 5713, 5714, 5715, 5716, 5717,5727, 5740, 5741, 5743, 5744, 5745, 5746, 5747, 5748, 5749, 5750, 5751,5755, 5757, 5758, 5759, 5760, 5761, 5762, 5763, 5764, 5765, 5766, 5767,5768, 5769, 5770, 5772, 5773, 5775, 5776, 5777, 5778, 5779, 5780, 5783,5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795,5796, 5797, 5798, 5799, 5800, 5801, 5802, 5803, 5811, 5812, 5813, 5814,5821, 5822, 5823, 5824, 5825, 5826, 5827, 5828, 5829, 5830, 5831, 5833,5834, 5836, 5837, 5838, 5839, 5848, 5849, 5850, 5851, 5852, 5853, 5854,5855, 5864, 5865, 5866, 5867, 5868, 5875, 5876, 5877, 5878, 5880, 5881,5882, 5883, 5884, 5885, 5886, 5887, 5888, 5889, 5890, 5891, 5892, 5893,5894, 5895, 5896, 5897, 5898, 5899, 5907, 5908, 5909, 5911, 5912, 5913,5914, 5915, 5916, 5917, 5918, 5919, 5920, 5921, 5922, 5929, 5950, 5951,5952, 5953, 5954, 5955, 5956, 5963, 5967, 5969, 5970, 5982, 5983, 5984,5985, 5986, 5987, 5988, 5997, 5998, 5999, 6000, 6001, 6002, 6068, 6069,6070, 6071, 6072, 6074, 6075, 6076, 6077, 6078, 6080, 6081, 6082, 6083,6084, 6085, 6086, 6087, 6088, 6089, 6090, 6091, 6092, 6093, 6094, 6095,6096, 6097, 6098, 6100, 6101, 6102, 6103, 6105, 6106, 6107, 6108, 6109,6110, 6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118, 6119, 6120, 6121,6122, 6123, 6124, 6128, 6130, 6142, 6143, 6144, 6145, 6146, 6147, 6148,6149, 6151, 6152, 6153, 6154, 6155, 6156, 6157, 6169, 6170, 6171, 6172,6173, 6174, 6175, 6176, 6177, 6178, 6179, 6180, 6181, 6182, 6185, 6186,6187, 6191, 6208, 6209, 6231, 6232, 6233, 6234, 6235, 6236, 6237, 6238,6239, 6251, 6256, 6257, 6258, 6259, 6260, 6261, 6262, 6278, 6279, 6280,6281, 6282, 6286, 6287, 6288, 6289, 6290, 6298, 6299, 6300, 6301, 6302,6303, 6304, 6305, 6306, 6307, 6308, 6309, 6310, 6311, 6312, 6313, 6314,6315, 6356, 6357, 6358, 6378, 6379, 6387, 6388, 6389, 6390, 6391, 6392,6393, 6394, 6395, 6396, 6397, 6398, 6399, 6400, 6401, 6402, 6403, 6405,6406, 6407, 6451, 6452, 6453, 6497, 6498, 6499, 6500, 6501, 6502, 6503,6509, 6510, 6511, 6512, 6514, 6519, 6520, 6521, 6522, 6523, 6549, 6550,6551, 6552, 6553, 6554, 6555, 6556, 6557, 6558, 6588, 6589, 6599, 6600,6601, 6602, 6603, 6604, 6605, 6606, 6611, 6612, 6613, 6614, 6615, 6616,6617, 6618, 6619, 6620, 6621, 6622, 6623, 6624, 6625, 6626, 6627, 6628,6629, 6630, 6631, 6632, 6633, 6634, 6638, 6639, 6640, 6641, 6642, 6643,6644, 6658, 6684, 6685, 6686, 6697, 6698, 6699, 6700, 6701, 6702, 6703,6704, 6705, 6706, 6707, 6708, 6709, 6710, 6719, 6721, 6723, 6724, 6754,6755, 6756, 6757, 6758, 6759, 6760, 6761, 6762, 6763, 6764, 6796, 6797,6798, 6799, 6800, 6805, 6806, 6807, 6808, 6809, 6814, 6815, 6816, 6817,6818, 6824, 6825, 6826, 6831, 6832, 6833, 6834, 6853, 6854, 6855, 6856,6857, 6860, 6861, 6862, 6863, 6864, 6865, 6866, 6867, 6868, 6869, 6872,6873, 6875, 6876, 6877, 6878, 6879, 6885, 6888, 6889, 6890, 6891, 6892,6893, 6894, 6895, 6896, 6897, 6898, 6899, 6900, 6901, 6902, 6903, 6904,6905, 6906, 6907, 6908, 6909, 6910, 6911, 6912, 6913, 6914, 6915, 6916,6917, 6918, 6919, 6920, 6921, 6922, 6923, 6924, 6925, 6926, 6927, 6928,6929, 6930, 6931, 6932, 6933, 6934, 6935, 6936, 6953, 6954, 6955, 6957,6958, 6959, 6960, 6962, 6963, 6964, 6965, 6966, 6967, 6968, 6969, 6970,6971, 6972, 6973, 6983, 6984, 6985, 6988, 6989, 6990, 6991, 6992, 6993,6994, 6995, 7007, 7008, 7009, 7010, 7011, 7012, 7015, 7016, 7017, 7018,7019, 7020, 7021, 7034, 7035, 7084, 7086, 7087, 7088, 7089, 7090, 7091,7092, 7128, 7137, 7138, 7139, 7140, 7141, 7142, 7143, 7144, 7145, 7146,7147, 7148, 7149, 7150, 7151, 7152, 7153, 7154, 7155, 7195, 7196, 7197,7198, 7200, 7201, 7202, 7203, 7204, 7205, 7206, 7207, 7208, 7209, 7210,7211, 7212, 7213, 7214, 7215, 7216, 7217, 7218, 7219, 7220, 7221, 7222,7223, 7224, 7225, 7226, 7227, 7228, 7229, 7230, 7231, 7232, 7233, 7234,7235, 7236, 7237, 7238, 7239, 7240, 7241, 7243, 7244, 7245, 7246, 7247,7248, 7249, 7250, 7251, 7252, 7253, 7254, 7255, 7256, 7257, 7258, 7259,7260, 7261, 7262, 7263, 7264, 7265, 7266, 7267, 7268, 7269, 7270, 7271,7291, 7292, 7293, 7294, 7295, 7296, 7297, 7298, 7305, 7306, 7307, 7308,7309, 7310, 7311, 7312, 7313, 7314, 7315, 7316, 7317, 7347, 7348, 7349,7358, 7359, 7360, 7361, 7362, 7363, 7364, 7365, 7372, 7373, 7374, 7375,7376, 7377, 7378, 7383, 7415, 7416, 7417, 7438, 7471, 7472, 7473, 7474,7475, 7476, 7477, 7490, 7496, 7497, 7498, 7499, 7500, 7501, 7502, 7503,7504, 7505, 7506, 7507, 7508, 7509, 7510, 7511, 7512, 7513, 7514, 7515,7532, 7533, 7534, 7535, 7537, 7538, 7539, 7540, 7541, 7542, 7543, 7544,7545, 7546, 7547, 7548, 7549, 7550, 7551, 7552, 7553, 7554, 7555, 7556,7557, 7558, 7559, 7560, 7585, 7586, 7587, 7588, 7589, 7590, 7591, 7592,7593, 7594, 7595, 7596, 7597, 7599, 7600, 7601, 7617, 7618, 7619, 7620,7621, 7622, 7623, 7638, 7668, 7669, 7670, 7672, 7673, 7674, 7675, 7676,7685, 7687, 7694, 7695, 7696, 7717, 7718, 7719, 7720, 7721, 7722, 7723,7724, 7725, 7726, 7727, 7728, 7729, 7730, 7731, 7732, 7733, 7734, 7735,7736, 7737, 7738, 7739, 7740, 7741, 7742, 7743, 7744, 7746, 7747, 7748,7749, 7750, 7751, 7752, 7757, 7758, 7759, 7760, 7761, 7762, 7763, 7764,7765, 7766, 7767, 7768, 7796, 7797, 7805, 7813, 7814, 7815, 7816, 7817,7818, 7819, 7820, 7821, 7822, 7848, 7849, 7850, 7851, 7852, 7853, 7854,7859, 7860, 7861, 7862, 7863, 7864, 7865, 7866, 7867, 7869, 7870, 7874,7875, 7917, 7918, 7919, 7925, 7926, 7927, 7928, 7929, 7941, 7942, 7943,7944, 7945, 7946, 7947, 7948, 7949, 7950, 7951, 7952, 7953, 7955, 7956,7957, 7958, 7959, 7960, 7961, 7962, 7963, 7964, 7965, 7966, 7967, 7968,7969, 7970, 7971, 7972, 7974, 7975, 7976, 7977, 7978, 7979, 7980, 7981,7982, 7983, 7984, 7985, 7986, 7987, 7988, 7989, 7990, 7991, 7992, 7993,7994, 7995, 7996, 7997, 7998, 7999, 8000, 8011, 8012, 8013, 8014, 8015,8016, 8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027,8028, 8029, 8030, 8048, 8049, 8050, 8051, 8052, 8053, 8054, 8055, 8056,8057, 8087, 8088, 8089, 8090, 8091, 8092, 8095, 8096, 8097, 8098, 8099,8100, 8101, 8102, 8103, 8104, 8105, 8106, 8107, 8110, 8111, 8112, 8113,8114, 8115, 8116, 8117, 8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125,8126, 8127, 8130, 8131, 8132, 8133, 8134, 8135, 8136, 8144, 8145, 8166,8169, 8170, 8171, 8172, 8173, 8174, 8175, 8176, 8177, 8178, 8179, 8180,8181, 8182, 8188, 8191, 8192, 8193, 8194, 8195, 8196, 8197, 8198, 8199,8200, 8201, 8209, 8210, 8211, 8212, 8215, 8227, 8230, 8231, 8232, 8233,8234, 8235, 8236, 8237, 8238, 8239, 8240, 8241, 8242, 8243, 8244, 8245,8246, 8247, 8248, 8249, 8250, 8251, 8260, 8261, 8262, 8263, 8299, 8302,8303, 8304, 8305, 8306, 8353, 8354, 8355, 8356, 8357, 8358, 8359, 8360,8361, 8375, 8376, 8377, 8378, 8379, 8380, 8381, 8382, 8410, 8411, 8412,8440, 8459, 8460, 8461, 8462, 8463, 8474, 8475, 8476, 8477, 8478, 8508,8509, 8510, 8511, 8512, 8514, 8515, 8546, 8547, 8548, 8549, 8550, 8551,8552, 8553, 8554, 8555, 8570, 8571, 8572, 8573, 8574, 8575, 8576, 8577,8578, 8579, 8580, 8581, 8586, 8587, 8593, 8594, 8595, 8596, 8597, 8601,8602, 8603, 8604, 8605, 8615, 8617, 8618, 8655, 8686, 8709, 8712, 8713,8714, 8715, 8716, 8717, 8718, 8719, 8720, 8721, 8722, 8723, 8724, 8725,8726, 8727, 8728, 8729, 8730, 8731, 8732, 8733, 8734, 8770, 8771, 8788,8789, 8790, 8791, 8792, 8793, 8794, 8795, 8796, 8797, 8798, 8799, 8800,8801, 8802, 8803, 8804, 8805, 8806, 8810, 8811, 8817, 8818, 8819, 8820,8821, 8822, 8823, 8829, 8830, 8837, 8838, 8839, 8840, 8841, 8848, 8849,8850, 8851, 8854, 8855, 8856, 8857, 8858, 8859, 8860, 8861, 8862, 8863,8864, 8865, 8867, 8868, 8872, 8873, 8874, 8875, 8876, 8878, 8879, 8880,8881, 8882, 8883, 8884, 8885, 8886, 8887, 8888, 8889, 8890, 8891, 8892,8893, 8894, 8895, 8896, 8897, 8898, 8899, 8900, 8901, 8902, 8903, 8904,8906, 8913, 8914, 8915, 8916, 8917, 8918, 8919, 8920, 8921, 8925, 8926,8927, 8928, 8933, 8934, 8935, 8936, 8937, 8938, 8939, 8940, 8941, 8942,8943, 8944, 8945, 8946, 8947, 8948, 8949, 8950, 8951, 8954, 8955, 8956,8992, 8993, 8994, 8995, 8996, 8997, 8998, 8999, 9000, 9001, 9002, 9003,9010, 9011, 9012, 9013, 9014, 9018, 9019, 9020, 9021, 9022, 9023, 9024,9025, 9026, 9027, 9028, 9029, 9030, 9031, 9041, 9042, 9043, 9044, 9045,9046, 9047, 9048, 9049, 9050, 9051, 9052, 9053, 9054, 9055, 9056, 9057,9058, 9059, 9065, 9066, 9087, 9088, 9091, 9092, 9093, 9094, 9095, 9096,9097, 9098, 9103, 9104, 9105, 9106, 9107, 9108, 9109, 9110, 9123, 9124,9125, 9126, 9127, 9128, 9129, 9130, 9131, 9133, 9134, 9135, 9136, 9137,9145, 9146, 9147, 9148, 9149, 9150, 9151, 9160, 9161, 9162, 9163, 9201,9203.

Avoidance of the Immune Response

As described herein, a useful feature of the oncology-relatedpolynucleotides, primary constructs or mmRNA of the invention is thecapacity to reduce, evade or avoid the innate immune response of a cell.In one aspect, provided herein are polynucleotides, primary constructsor mmRNA encoding a polypeptide of interest which when delivered tocells, results in a reduced immune response from the host as compared tothe response triggered by a reference compound, e.g. an unmodifiedpolynucleotide corresponding to a polynucleotide, primary construct ormmRNA of the invention, or a different polynucleotide, primary constructor mmRNA of the invention. As used herein, a “reference compound” is anymolecule or substance which when administered to a mammal, results in aninnate immune response having a known degree, level or amount of immunestimulation. A reference compound need not be a nucleic acid moleculeand it need not be any of the oncology-related polynucleotides, primaryconstructs or mmRNA of the invention. Hence, the measure of apolynucleotides, primary constructs or mmRNA avoidance, evasion orfailure to trigger an immune response can be expressed in terms relativeto any compound or substance which is known to trigger such a response.

The term “innate immune response” includes a cellular response toexogenous single stranded nucleic acids, generally of viral or bacterialorigin, which involves the induction of cytokine expression and release,particularly the interferons, and cell death. As used herein, the innateimmune response or interferon response operates at the single cell levelcausing cytokine expression, cytokine release, global inhibition ofprotein synthesis, global destruction of cellular RNA, upregulation ofmajor histocompatibility molecules, and/or induction of apoptotic death,induction of gene transcription of genes involved in apoptosis,anti-growth, and innate and adaptive immune cell activation. Some of thegenes induced by type I IFNs include PKR, ADAR (adenosine deaminaseacting on RNA), OAS (2′,5′-oligoadenylate synthetase), RNase L, and Mxproteins. PKR and ADAR lead to inhibition of translation initiation andRNA editing, respectively. OAS is a dsRNA-dependent synthetase thatactivates the endoribonuclease RNase L to degrade ssRNA.

In some embodiments, the innate immune response comprises expression ofa Type I or Type II interferon, and the expression of the Type I or TypeII interferon is not increased more than two-fold compared to areference from a cell which has not been contacted with apolynucleotide, primary construct or mmRNA of the invention.

In some embodiments, the innate immune response comprises expression ofone or more IFN signature genes and where the expression of the one ofmore IFN signature genes is not increased more than three-fold comparedto a reference from a cell which has not been contacted with thepolynucleotide, primary construct or mmRNA of the invention.

While in some circumstances, it might be advantageous to eliminate theinnate immune response in a cell, the invention providespolynucleotides, primary constructs and mmRNA that upon administrationresult in a substantially reduced (significantly less) the immuneresponse, including interferon signaling, without entirely eliminatingsuch a response.

In some embodiments, the immune response is lower by 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% ascompared to the immune response induced by a reference compound. Theimmune response itself may be measured by determining the expression oractivity level of Type 1 interferons or the expression ofinterferon-regulated genes such as the toll-like receptors (e.g., TLR7and TLR8). Reduction of innate immune response can also be measured bymeasuring the level of decreased cell death following one or moreadministrations to a cell population; e.g., cell death is 10%, 25%, 50%,75%, 85%, 90%, 95%, or over 95% less than the cell death frequencyobserved with a reference compound. Moreover, cell death may affectfewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than0.01% of cells contacted with the polynucleotide, primary construct ormmRNA.

In another embodiment, the polynucleotide, primary construct or mmRNA ofthe present invention is significantly less immunogenic than anunmodified in vitro-synthesized RNA molecule polynucleotide, or primaryconstruct with the same sequence or a reference compound. As usedherein, “significantly less immunogenic” refers to a detectable decreasein immunogenicity. In another embodiment, the term refers to a folddecrease in immunogenicity. In another embodiment, the term refers to adecrease such that an effective amount of the polynucleotide, primaryconstruct or mmRNA can be administered without triggering a detectableimmune response. In another embodiment, the term refers to a decreasesuch that the polynucleotide, primary construct or mmRNA can berepeatedly administered without eliciting an immune response sufficientto detectably reduce expression of the recombinant protein. In anotherembodiment, the decrease is such that the polynucleotide, primaryconstruct or mmRNA can be repeatedly administered without eliciting animmune response sufficient to eliminate detectable expression of therecombinant protein.

In another embodiment, the polynucleotide, primary construct or mmRNA is2-fold less immunogenic than its unmodified counterpart or referencecompound. In another embodiment, immunogenicity is reduced by a 3-foldfactor. In another embodiment, immunogenicity is reduced by a 5-foldfactor. In another embodiment, immunogenicity is reduced by a 7-foldfactor. In another embodiment, immunogenicity is reduced by a 10-foldfactor. In another embodiment, immunogenicity is reduced by a 15-foldfactor. In another embodiment, immunogenicity is reduced by a foldfactor. In another embodiment, immunogenicity is reduced by a 50-foldfactor. In another embodiment, immunogenicity is reduced by a 100-foldfactor. In another embodiment, immunogenicity is reduced by a 200-foldfactor. In another embodiment, immunogenicity is reduced by a 500-foldfactor. In another embodiment, immunogenicity is reduced by a 1000-foldfactor. In another embodiment, immunogenicity is reduced by a 2000-foldfactor. In another embodiment, immunogenicity is reduced by another folddifference.

Methods of determining immunogenicity are well known in the art, andinclude, e.g. measuring secretion of cytokines (e.g. IL-12, IFNalpha,TNF-alpha, RANTES, MIP-1alpha or beta, IL-6, IFN-beta, or IL-8),measuring expression of DC activation markers (e.g. CD83, HLA-DR, CD80and CD86), or measuring ability to act as an adjuvant for an adaptiveimmune response.

The polynucleotide, primary construct or mmRNA of the invention,including the combination of modifications taught herein may havesuperior properties making them more suitable as therapeutic modalities.

It has been determined that the “all or none” model in the art is sorelyinsufficient to describe the biological phenomena associated with thetherapeutic utility of modified mRNA. The present inventors havedetermined that to improve protein production, one may consider thenature of the modification, or combination of modifications, the percentmodification and survey more than one cytokine or metric to determinethe efficacy and risk profile of a particular modified mRNA.

In one aspect of the invention, methods of determining the effectivenessof a modified mRNA as compared to unmodified involves the measure andanalysis of one or more cytokines whose expression is triggered by theadministration of the exogenous nucleic acid of the invention. Thesevalues are compared to administration of an unmodified nucleic acid orto a standard metric such as cytokine response, PolyIC, R-848 or otherstandard known in the art.

One example of a standard metric developed herein is the measure of theratio of the level or amount of encoded polypeptide (protein) producedin the cell, tissue or organism to the level or amount of one or more(or a panel) of cytokines whose expression is triggered in the cell,tissue or organism as a result of administration or contact with themodified nucleic acid. Such ratios are referred to herein as theProtein:Cytokine Ratio or “PC” Ratio. The higher the PC ratio, the moreefficacious the modified nucleic acid (polynucleotide encoding theprotein measured). Preferred PC Ratios, by cytokine, of the presentinvention may be greater than 1, greater than 10, greater than 100,greater than 1000, greater than 10,000 or more. Modified nucleic acidshaving higher PC Ratios than a modified nucleic acid of a different orunmodified construct are preferred.

The PC ratio may be further qualified by the percent modificationpresent in the polynucleotide. For example, normalized to a 100%modified nucleic acid, the protein production as a function of cytokine(or risk) or cytokine profile can be determined.

In one embodiment, the present invention provides a method fordetermining, across chemistries, cytokines or percent modification, therelative efficacy of any particular modified the polynucleotide, primaryconstruct or mmRNA by comparing the PC Ratio of the modified nucleicacid (polynucleotide, primary construct or mmRNA).

mmRNA containing varying levels of nucleobase substitutions could beproduced that maintain increased protein production and decreasedimmunostimulatory potential. The relative percentage of any modifiednucleotide to its naturally occurring nucleotide counterpart can bevaried during the IVT reaction (for instance, 100, 50, 25, 10, 5, 2.5,1, 0.1, 0.01% 5 methyl cytidine usage versus cytidine; 100, 50, 25, 10,5, 2.5, 1, 0.1, 0.01% pseudouridine or N1-methyl-pseudouridine usageversus uridine). mmRNA can also be made that utilize different ratiosusing 2 or more different nucleotides to the same base (for instance,different ratios of pseudouridine and N1-methyl-pseudouridine). mmRNAcan also be made with mixed ratios at more than 1 “base” position, suchas ratios of 5 methyl cytidine/cytidine andpseudouridine/N1-methyl-pseudouridine/uridine at the same time. Use ofmodified mRNA with altered ratios of modified nucleotides can bebeneficial in reducing potential exposure to chemically modifiednucleotides. Lastly, positional introduction of modified nucleotidesinto the mmRNA which modulate either protein production orimmunostimulatory potential or both is also possible. The ability ofsuch mmRNA to demonstrate these improved properties can be assessed invitro (using assays such as the PBMC assay described herein), and canalso be assessed in vivo through measurement of both mmRNA-encodedprotein production and mediators of innate immune recognition such ascytokines.

In another embodiment, the relative immunogenicity of thepolynucleotide, primary construct or mmRNA and its unmodifiedcounterpart are determined by determining the quantity of thepolynucleotide, primary construct or mmRNA required to elicit one of theabove responses to the same degree as a given quantity of the unmodifiednucleotide or reference compound. For example, if twice as muchpolynucleotide, primary construct or mmRNA is required to elicit thesame response, than the polynucleotide, primary construct or mmRNA istwo-fold less immunogenic than the unmodified nucleotide or thereference compound.

In another embodiment, the relative immunogenicity of thepolynucleotide, primary construct or mmRNA and its unmodifiedcounterpart are determined by determining the quantity of cytokine (e.g.IL-12, IFNalpha, TNF-alpha, RANTES, MIP-1alpha or beta, IL-6, IFN-beta,or IL-8) secreted in response to administration of the polynucleotide,primary construct or mmRNA, relative to the same quantity of theunmodified nucleotide or reference compound. For example, if one-half asmuch cytokine is secreted, than the polynucleotide, primary construct ormmRNA is two-fold less immunogenic than the unmodified nucleotide. Inanother embodiment, background levels of stimulation are subtractedbefore calculating the immunogenicity in the above methods.

Provided herein are also methods for performing the titration, reductionor elimination of the immune response in a cell or a population ofcells. In some embodiments, the cell is contacted with varied doses ofthe same polynucleotides, primary constructs or mmRNA and dose responseis evaluated. In some embodiments, a cell is contacted with a number ofdifferent polynucleotides, primary constructs or mmRNA at the same ordifferent doses to determine the optimal composition for producing thedesired effect. Regarding the immune response, the desired effect may beto avoid, evade or reduce the immune response of the cell. The desiredeffect may also be to alter the efficiency of protein production.

The polynucleotides, primary constructs and/or mmRNA of the presentinvention may be used to reduce the immune response using the methoddescribed in International Publication No. WO2013003475, hereinincorporated by reference in its entirety.

Activation of the Immune Response: Vaccines

Additionally, certain modified nucleosides, or combinations thereof,when introduced into the oncology-related polynucleotides, primaryconstructs or mmRNA of the invention will activate the innate immuneresponse. Such activating molecules are useful as adjuvants whencombined with polypeptides and/or other vaccines. In certainembodiments, the activating molecules contain a translatable regionwhich encodes for a polypeptide sequence useful as a vaccine, thusproviding the ability to be a self-adjuvant.

In one embodiment, the oncology-related polynucleotides, primaryconstructs and/or mmRNA of the invention may encode an immunogen. Thedelivery of the oncology-related polynucleotides, primary constructsand/or mmRNA encoding an immunogen may activate the immune response. Asa non-limiting example, the oncology-related polynucleotides, primaryconstructs and/or mmRNA encoding an immunogen may be delivered to cellsto trigger multiple innate response pathways (see International Pub. No.WO2012006377; herein incorporated by reference in its entirety). Asanother non-limiting example, the oncology-related polynucleotides,primary constructs and mmRNA of the present invention encoding animmunogen may be delivered to a vertebrate in a dose amount large enoughto be immunogenic to the vertebrate (see International Pub. Nos.WO2012006372 and WO2012006369; each of which is herein incorporated byreference in their entirety).

The oncology-related polynucleotides, primary constructs or mmRNA ofinvention may encode a polypeptide sequence for a vaccine and mayfurther comprise an inhibitor. The inhibitor may impair antigenpresentation and/or inhibit various pathways known in the art. As anon-limiting example, the oncology-related polynucleotides, primaryconstructs or mmRNA of the invention may be used for a vaccine incombination with an inhibitor which can impair antigen presentation (seeInternational Pub. No. WO2012089225 and WO2012089338; each of which isherein incorporated by reference in their entirety).

In one embodiment, the oncology-related polynucleotides, primaryconstructs or mmRNA of the invention may be self-replicating RNA.Self-replicating RNA molecules can enhance efficiency of RNA deliveryand expression of the enclosed gene product. In one embodiment, theoncology-related polynucleotides, primary constructs or mmRNA maycomprise at least one modification described herein and/or known in theart. In one embodiment, the self-replicating RNA can be designed so thatthe self-replicating RNA does not induce production of infectious viralparticles. As a non-limiting example the self-replicating RNA may bedesigned by the methods described in US Pub. No. US20110300205 andInternational Pub. No. WO2011005799, each of which is hereinincorporated by reference in their entirety.

In one embodiment, the self-replicating polynucleotides, primaryconstructs or mmRNA of the invention may encode a protein which mayraise the immune response. As a non-limiting example, theoncology-related polynucleotides, primary constructs or mmRNA may beself-replicating mRNA may encode at least one antigen (see US Pub. No.US20110300205 and International Pub. Nos. WO2011005799, WO2013006838 andWO2013006842; each of which is herein incorporated by reference in theirentirety).

In one embodiment, the self-replicating polynucleotides, primaryconstructs or mmRNA of the invention may be formulated using methodsdescribed herein or known in the art. As a non-limiting example, theself-replicating RNA may be formulated for delivery by the methodsdescribed in Geall et al (Nonviral delivery of self-amplifying RNAvaccines, PNAS 2012; PMID: 22908294).

In one embodiment, the oncology-related polynucleotides, primaryconstructs or mmRNA of the present invention may encode amphipathicand/or immunogenic amphipathic peptides.

In on embodiment, a formulation of the oncology-related polynucleotides,primary constructs or mmRNA of the present invention may furthercomprise an amphipathic and/or immunogenic amphipathic peptide. As anon-limiting example, the oncology-related polynucleotides, primaryconstructs or mmRNA comprising an amphipathic and/or immunogenicamphipathic peptide may be formulated as described in US. Pub. No.US20110250237 and International Pub. Nos. WO2010009277 and WO2010009065;each of which is herein incorporated by reference in their entirety.

In one embodiment, the oncology-related polynucleotides, primaryconstructs or mmRNA of the present invention may be immunostimulatory.As a non-limiting example, the oncology-related polynucleotides, primaryconstructs or mmRNA may encode all or a part of a positive-sense or anegative-sense stranded RNA virus genome (see International Pub No.WO2012092569 and US Pub No. US20120177701, each of which is hereinincorporated by reference in their entirety). In another non-limitingexample, the immunostimulatory polynucleotides, primary constructs ormmRNA of the present invention may be formulated with an excipient foradministration as described herein and/or known in the art (seeInternational Pub No. WO2012068295 and US Pub No. US20120213812, each ofwhich is herein incorporated by reference in their entirety).

In one embodiment, the response of the vaccine formulated by the methodsdescribed herein may be enhanced by the addition of various compounds toinduce the therapeutic effect. As a non-limiting example, the vaccineformulation may include a MHC II binding peptide or a peptide having asimilar sequence to a MHC II binding peptide (see International Pub Nos.WO2012027365, WO2011031298 and US Pub No. US20120070493, US20110110965,each of which is herein incorporated by reference in their entirety). Asanother example, the vaccine formulations may comprise modifiednicotinic compounds which may generate an antibody response to nicotineresidue in a subject (see International Pub No. WO2012061717 and US PubNo. US20120114677, each of which is herein incorporated by reference intheir entirety).

Naturally Occurring Mutants

In another embodiment, the oncology-related polynucleotides, primaryconstruct and/or mmRNA can be utilized to express variants of naturallyoccurring proteins that have an improved disease modifying activity,including increased biological activity, improved patient outcomes, or aprotective function, etc. Many such modifier genes have been describedin mammals (Nadeau, Current Opinion in Genetics & Development 200313:290-295; Hamilton and Yu, PLoS Genet. 2012; 8:e1002644; Corder etal., Nature Genetics 1994 7:180-184; all herein incorporated byreference in their entireties). Examples in humans include Apo E2protein, Apo A-I variant proteins (Apo A-I Milano, Apo A-I Paris),hyperactive Factor IX protein (Factor IX Padua Arg338Lys), transthyretinmutants (TTR Thr119Met). Expression of ApoE2 (cys112, cys158) has beenshown to confer protection relative to other ApoE isoforms (ApoE3(cys112, arg158), and ApoE4 (arg112, arg158)) by reducing susceptibilityto Alzheimer's disease and possibly other conditions such ascardiovascular disease (Corder et al., Nature Genetics 1994 7:180-184;Seripa et al., Rejuvenation Res. 2011 14:491-500; Liu et al. Nat RevNeurol. 2013 9:106-118; all herein incorporated by reference in theirentireties). Expression of Apo A-I variants has been associated withreduced cholesterol (deGoma and Rader, 2011 Nature Rev Cardiol8:266-271; Nissen et al., 2003 JAMA 290:2292-2300; all hereinincorporated by reference in its entirety). The amino acid sequence ofApoA-I in certain populations has been changed to cysteine in Apo A-IMilano (Arg 173 changed to Cys) and in Apo A-I Paris (Arg 151 changed toCys). Factor IX mutation at position R338L (FIX Padua) results in aFactor IX protein that has ˜10-fold increased activity (Simioni et al.,N Engl J Med. 2009 361:1671-1675; Finn et al., Blood. 2012120:4521-4523; Cantore et al., Blood. 2012 120:4517-20; all hereinincorporated by reference in their entireties). Mutation oftransthyretin at positions 104 or 119 (Arg104 His, Thr119Met) has beenshown to provide protection to patients also harboring the diseasecausing Val30Met mutations (Saraiva, Hum Mutat. 2001 17:493-503; DATABASE ON TRANSTHYRETIN MUTATIONS www.ibmc.up.pt/mjsaraiva/ttrmut.html;all herein incorporated by reference in its entirety). Differences inclinical presentation and severity of symptoms among Portuguese andJapanese Met 30 patients carrying respectively the Met 119 and theHis104 mutations are observed with a clear protective effect exerted bythe non pathogenic mutant (Coelho et al. 1996 Neuromuscular Disorders(Suppl) 6: S20; Terazaki et al. 1999. Biochem Biophys Res Commun 264:365-370; all herein incorporated by reference in its entirety), whichconfer more stability to the molecule. A modified mRNA encoding theseprotective TTR alleles can be expressed in TTR amyloidosis patients,thereby reducing the effect of the pathogenic mutant TTR protein.

Major Groove Interacting Partners

As described herein, the phrase “major groove interacting partner”refers to RNA recognition receptors that detect and respond to RNAligands through interactions, e.g. binding, with the major groove faceof a nucleotide or nucleic acid. As such, RNA ligands comprisingmodified nucleotides or nucleic acids such as the polynucleotide,primary construct or mmRNA as described herein decrease interactionswith major groove binding partners, and therefore decrease an innateimmune response.

Example major groove interacting, e.g. binding, partners include, butare not limited to the following nucleases and helicases. Withinmembranes, TLRs (Toll-like Receptors) 3, 7, and 8 can respond to single-and double-stranded RNAs. Within the cytoplasm, members of thesuperfamily 2 class of DEX(D/H) helicases and ATPases can sense RNAs toinitiate antiviral responses. These helicases include the RIG-I(retinoic acid-inducible gene I) and MIDAS(melanomadifferentiation-associated gene 5). Other examples include laboratory ofgenetics and physiology 2 (LGP2), HIN-200 domain containing proteins, orHelicase-domain containing proteins.

Targeting of Pathogenic Organisms or Diseased Cells

Provided herein are methods for targeting pathogenic microorganisms,such as bacteria, yeast, protozoa, helminthes and the like, or diseasedcells such as cancer cells using polynucleotides, primary constructs ormmRNA that encode cytostatic or cytotoxic polypeptides. Preferably themRNA introduced contains modified nucleosides or other nucleic acidsequence modifications that are translated exclusively, orpreferentially, in the target pathogenic organism, to reduce possibleoff-target effects of the therapeutic. Such methods are useful forremoving pathogenic organisms or killing diseased cells found in anybiological material, including blood, semen, eggs, and transplantmaterials including embryos, tissues, and organs.

Bioprocessing

The methods provided herein may be useful for enhancing protein productyield in a cell culture process. In a cell culture containing aplurality of host cells, introduction of a polynucleotide, primaryconstruct or mmRNA described herein results in increased proteinproduction efficiency relative to a corresponding unmodified nucleicacid. Such increased protein production efficiency can be demonstrated,e.g., by showing increased cell transfection, increased proteintranslation from the polynucleotide, primary construct or mmRNA,decreased nucleic acid degradation, and/or reduced innate immuneresponse of the host cell. Protein production can be measured byenzyme-linked immunosorbent assay (ELISA), and protein activity can bemeasured by various functional assays known in the art. The proteinproduction may be generated in a continuous or a batch-fed mammalianprocess.

Additionally, it is useful to optimize the expression of a specificpolypeptide in a cell line or collection of cell lines of potentialinterest, particularly a polypeptide of interest such as a proteinvariant of a reference protein having a known activity. In oneembodiment, provided is a method of optimizing expression of apolypeptide of interest in a target cell, by providing a plurality oftarget cell types, and independently contacting with each of theplurality of target cell types a polynucleotide, primary construct ormmRNA encoding a polypeptide of interest. The cells may be transfectedwith two or more polynucleotide, primary construct or mmRNAsimultaneously or sequentially.

In certain embodiments, multiple rounds of the methods described hereinmay be used to obtain cells with increased expression of one or morenucleic acids or proteins of interest. For example, cells may betransfected with one or more polynucleotide, primary construct or mmRNAthat encode a nucleic acid or protein of interest. The cells may beisolated according to methods described herein before being subjected tofurther rounds of transfections with one or more other nucleic acidswhich encode a nucleic acid or protein of interest before being isolatedagain. This method may be useful for generating cells with increasedexpression of a complex of proteins, nucleic acids or proteins in thesame or related biological pathway, nucleic acids or proteins that actupstream or downstream of each other, nucleic acids or proteins thathave a modulating, activating or repressing function to each other,nucleic acids or proteins that are dependent on each other for functionor activity, or nucleic acids or proteins that share homology.

Additionally, culture conditions may be altered to increase proteinproduction efficiency. Subsequently, the presence and/or level of thepolypeptide of interest in the plurality of target cell types isdetected and/or quantitated, allowing for the optimization of apolypeptide's expression by selection of an efficient target cell andcell culture conditions relating thereto. Such methods are particularlyuseful when the polypeptide contains one or more post-translationalmodifications or has substantial tertiary structure, situations whichoften complicate efficient protein production.

In one embodiment, the cells used in the methods of the presentinvention may be cultured. The cells may be cultured in suspension or asadherent cultures. The cells may be cultured in a varied of vesselsincluding, but not limited to, bioreactors, cell bags, wave bags,culture plates, flasks and other vessels well known to those of ordinaryskill in the art. Cells may be cultured in IMDM (Invitrogen, Catalognumber 12440-53) or any other suitable media including, but not limitedto, chemically defined media formulations. The ambient conditions whichmay be suitable for cell culture, such as temperature and atmosphericcomposition, are well known to those skilled in the art. The methods ofthe invention may be used with any cell that is suitable for use inprotein production.

The invention provides for the repeated introduction (e.g.,transfection) of modified nucleic acids into a target cell population,e.g., in vitro, ex vivo, in situ, or in vivo. For example, contactingthe same cell population may be repeated one or more times (such as two,three, four, five or more than five times). In some embodiments, thestep of contacting the cell population with the oncology-relatedpolynucleotides, primary constructs or mmRNA is repeated a number oftimes sufficient such that a predetermined efficiency of proteintranslation in the cell population is achieved. Given the often reducedcytotoxicity of the target cell population provided by the nucleic acidmodifications, repeated transfections are achievable in a diverse arrayof cell types and within a variety of tissues, as provided herein.

In one embodiment, the bioprocessing methods of the present inventionmay be used to produce antibodies or functional fragments thereof. Thefunctional fragments may comprise a Fab, Fab′, F(ab′)2, an Fv domain, anscFv, or a diabody. They may be variable in any region including thecomplement determining region (CDR). In one embodiment, there iscomplete diversity in the CDR3 region. In another embodiment, theantibody is substantially conserved except in the CDR3 region.

Antibodies may be made which bind or associate with any biomolecule,whether human, pathogenic or non-human in origin. The pathogen may bepresent in a non-human mammal, a clinical specimen or from a commercialproduct such as a oncology-related or pharmaceutical material. They mayalso bind to any specimen or sample including clinical specimens ortissue samples from any organism.

In some embodiments, the contacting step is repeated multiple times at afrequency selected from the group consisting of: 6 hour, 12 hour, 24hour, 36 hour, 48 hour, 72 hour, 84 hour, 96 hour, and 108 hour and atconcentrations of less than 20 nM, less than 50 nM, less than 80 nM orless than 100 nM. Compositions may also be administered at less than 1mM, less than 5 mM, less than 10 mM, less than 100 mM or less than 500mM.

In some embodiments, the oncology-related polynucleotides, primaryconstructs or mmRNA are added at an amount of 50 molecules per cell, 100molecules/cell, 200 molecules/cell, 300 molecules/cell, 400molecules/cell, 500 molecules/cell, 600 molecules/cell, 700molecules/cell, 800 molecules/cell, 900 molecules/cell, 1000molecules/cell, 2000 molecules/cell, or 5000 molecules/cell.

In other embodiments, the oncology-related polynucleotides, primaryconstructs or mmRNA are added at a concentration selected from the groupconsisting of: 0.01 fmol/106 cells, 0.1 fmol/106 cells, 0.5 fmol/106cells, 0.75 fmol/106 cells, 1 fmol/106 cells, 2 fmol/106 cells, 5fmol/106 cells, 10 fmol/106 cells, 20 fmol/106 cells, 30 fmol/106 cells,40 fmol/106 cells, 50 fmol/106 cells, 60 fmol/106 cells, 100 fmol/106cells, 200 fmol/106 cells, 300 fmol/106 cells, 400 fmol/106 cells, 500fmol/106 cells, 700 fmol/106 cells, 800 fmol/106 cells, 900 fmol/106cells, and 1 pmol/106 cells.

In some embodiments, the production of a biological product upon isdetected by monitoring one or more measurable bioprocess parameters,such as a parameter selected from the group consisting of: cell density,pH, oxygen levels, glucose levels, lactic acid levels, temperature, andprotein production. Protein production can be measured as specificproductivity (SP) (the concentration of a product, such as aheterologously expressed polypeptide, in solution) and can be expressedas mg/L or g/L; in the alternative, specific productivity can beexpressed as pg/cell/day. An increase in SP can refer to an absolute orrelative increase in the concentration of a product produced under twodefined set of conditions (e.g., when compared with controls not treatedwith modified mRNA(s)).

Cells

In one embodiment, the cells are selected from the group consisting ofmammalian cells, bacterial cells, plant, microbial, algal and fungalcells. In some embodiments, the cells are mammalian cells, such as, butnot limited to, human, mouse, rat, goat, horse, rabbit, hamster or cowcells. In a further embodiment, the cells may be from an establishedcell line, including, but not limited to, HeLa, NSO, SP2/0, KEK 293T,Vero, Caco, Caco-2, MDCK, COS-1, COS-7, K562, Jurkat, CHO-K1, DG44,CHOK1SV, CHO—S, Huvec, CV-1, Huh-7, NIH3T3, HEK293, 293, A549, HepG2,IMR-90, MCF-7, U-20S, Per.C6, SF9, SF21 or Chinese Hamster Ovary (CHO)cells.

In certain embodiments, the cells are fungal cells, such as, but notlimited to, Chrysosporium cells, Aspergillus cells, Trichoderma cells,Dictyostelium cells, Candida cells, Saccharomyces cells,Schizosaccharomyces cells, and Penicillium cells.

In certain embodiments, the cells are bacterial cells such as, but notlimited to, E. coli, B. subtilis, or BL21 cells. Primary and secondarycells to be transfected by the methods of the invention can be obtainedfrom a variety of tissues and include, but are not limited to, all celltypes which can be maintained in culture. For examples, primary andsecondary cells which can be transfected by the methods of the inventioninclude, but are not limited to, fibroblasts, keratinocytes, epithelialcells (e.g., mammary epithelial cells, intestinal epithelial cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), muscle cells andprecursors of these somatic cell types. Primary cells may also beobtained from a donor of the same species or from another species (e.g.,mouse, rat, rabbit, cat, dog, pig, cow, bird, sheep, goat, horse).

Purification and Isolation

Those of ordinary skill in the art should be able to make adetermination of the methods to use to purify or isolate of a protein ofinterest from cultured cells. Generally, this is done through a capturemethod using affinity binding or non-affinity purification. If theprotein of interest is not secreted by the cultured cells, then a lysisof the cultured cells should be performed prior to purification orisolation. One may use unclarified cell culture fluid containing theprotein of interest along with cell culture media components as well ascell culture additives, such as anti-foam compounds and other nutrientsand supplements, cells, cellular debris, host cell proteins, DNA,viruses and the like in the present invention. The process may beconducted in the bioreactor itself. The fluid may either bepreconditioned to a desired stimulus such as pH, temperature or otherstimulus characteristic or the fluid can be conditioned upon theaddition of polymer(s) or the polymer(s) can be added to a carrierliquid that is properly conditioned to the required parameter for thestimulus condition required for that polymer to be solubilized in thefluid. The polymer may be allowed to circulate thoroughly with the fluidand then the stimulus may be applied (change in pH, temperature, saltconcentration, etc) and the desired protein and polymer(s) precipitatecan out of the solution. The polymer and the desired protein(s) can beseparated from the rest of the fluid and optionally washed one or moretimes to remove any trapped or loosely bound contaminants. The desiredprotein may then be recovered from the polymer(s) by, for example,elution and the like. Preferably, the elution may be done under a set ofconditions such that the polymer remains in its precipitated form andretains any impurities to it during the selected elution of the desiredprotein. The polymer and protein as well as any impurities may besolubilized in a new fluid such as water or a buffered solution and theprotein may be recovered by a means such as affinity, ion exchanged,hydrophobic, or some other type of chromatography that has a preferenceand selectivity for the protein over that of the polymer or impurities.The eluted protein may then be recovered and may be subjected toadditional processing steps, either batch like steps or continuous flowthrough steps if appropriate.

In another embodiment, it may be useful to optimize the expression of aspecific polypeptide in a cell line or collection of cell lines ofpotential interest, particularly a polypeptide of interest such as aprotein variant of a reference protein having a known activity. In oneembodiment, provided is a method of optimizing expression of apolypeptide of interest in a target cell, by providing a plurality oftarget cell types, and independently contacting with each of theplurality of target cell types a modified mRNA encoding a polypeptide.Additionally, culture conditions may be altered to increase proteinproduction efficiency. Subsequently, the presence and/or level of thepolypeptide of interest in the plurality of target cell types isdetected and/or quantitated, allowing for the optimization of apolypeptide of interest's expression by selection of an efficient targetcell and cell culture conditions relating thereto. Such methods may beuseful when the polypeptide of interest contains one or morepost-translational modifications or has substantial tertiary structure,which often complicate efficient protein production.

Protein Recovery

The protein of interest may be preferably recovered from the culturemedium as a secreted polypeptide, or it can be recovered from host celllysates if expressed without a secretory signal. It may be necessary topurify the protein of interest from other recombinant proteins and hostcell proteins in a way that substantially homogenous preparations of theprotein of interest are obtained. The cells and/or particulate celldebris may be removed from the culture medium or lysate. The product ofinterest may then be purified from contaminant soluble proteins,polypeptides and nucleic acids by, for example, fractionation onimmunoaffinity or ion-exchange columns, ethanol precipitation, reversephase HPLC (RP-HPLC), SEPHADEX® chromatography, chromatography on silicaor on a cation exchange resin such as DEAE. Methods of purifying aprotein heterologous expressed by a host cell are well known in the art.

Methods and compositions described herein may be used to produceproteins which are capable of attenuating or blocking the endogenousagonist biological response and/or antagonizing a receptor or signalingmolecule in a mammalian subject. For example, IL-12 and IL-23 receptorsignaling may be enhanced in chronic autoimmune disorders such asmultiple sclerosis and inflammatory diseases such as rheumatoidarthritis, psoriasis, lupus erythematosus, ankylosing spondylitis andChron's disease (Kikly K, Liu L, Na S, Sedgwich J D (2006) Cur. Opin.Immunol. 18(6): 670-5). In another embodiment, a nucleic acid encodes anantagonist for chemokine receptors. Chemokine receptors CXCR-4 and CCR-5are required for HIV enry into host cells (Arenzana-Seisdedos F et al,(1996) Nature. October 3; 383 (6599):400).

Gene Silencing

The oncology-related polynucleotides, primary constructs and mmRNAdescribed herein are useful to silence (i.e., prevent or substantiallyreduce) expression of one or more target genes in a cell population. Apolynucleotide, primary construct or mmRNA encoding a polypeptide ofinterest capable of directing sequence-specific histone H3 methylationis introduced into the cells in the population under conditions suchthat the polypeptide is translated and reduces gene transcription of atarget gene via histone H3 methylation and subsequent heterochromatinformation. In some embodiments, the silencing mechanism is performed ona cell population present in a mammalian subject. By way of non-limitingexample, a useful target gene is a mutated Janus Kinase-2 family member,wherein the mammalian subject expresses the mutant target gene suffersfrom a myeloproliferative disease resulting from aberrant kinaseactivity.

Co-administration of oncology-related polynucleotides, primaryconstructs and mmRNA and RNAi agents are also provided herein.

Modulation of Biological Pathways

The rapid translation polynucleotides, primary constructs and mmRNAintroduced into cells provides a desirable mechanism of modulatingtarget biological pathways. Such modulation includes antagonism oragonism of a given pathway. In one embodiment, a method is provided forantagonizing a biological pathway in a cell by contacting the cell withan effective amount of a composition comprising a polynucleotide,primary construct or mmRNA encoding a polypeptide of interest, underconditions such that the oncology-related polynucleotides, primaryconstructs and mmRNA is localized into the cell and the polypeptide iscapable of being translated in the cell from the oncology-relatedpolynucleotides, primary constructs and mmRNA, wherein the polypeptideinhibits the activity of a polypeptide functional in the biologicalpathway. Exemplary biological pathways are those defective in anautoimmune or inflammatory disorder such as multiple sclerosis,rheumatoid arthritis, psoriasis, lupus erythematosus, ankylosingspondylitis colitis, or Crohn's disease; in particular, antagonism ofthe IL-12 and IL-23 signaling pathways are of particular utility. (SeeKikly K, Liu L, Na S, Sedgwick J D (2006) Curr. Opin. Immunol. 18 (6):670-5; herein incorporated by reference in its entirety).

Further, provided are oncology-related polynucleotide, primary constructor mmRNA encoding an antagonist for chemokine receptors; chemokinereceptors CXCR-4 and CCR-5 are required for, e.g., HIV entry into hostcells (Arenzana-Seisdedos F et al, (1996) Nature. October 3;383(6599):400; herein incorporated by reference in its entirety).

Alternatively, provided are methods of agonizing a biological pathway ina cell by contacting the cell with an effective amount of anoncology-related polynucleotide, primary construct or mmRNA encoding arecombinant polypeptide under conditions such that the nucleic acid islocalized into the cell and the recombinant polypeptide is capable ofbeing translated in the cell from the nucleic acid, and the recombinantpolypeptide induces the activity of a polypeptide functional in thebiological pathway. Exemplary agonized biological pathways includepathways that modulate cell fate determination. Such agonization isreversible or, alternatively, irreversible.

Expression of Ligand or Receptor on Cell Surface

In some aspects and embodiments of the aspects described herein, theoncology-related polynucleotides, primary constructs or mmRNA describedherein can be used to express a ligand or ligand receptor on the surfaceof a cell (e.g., a homing moiety). A ligand or ligand receptor moietyattached to a cell surface can permit the cell to have a desiredbiological interaction with a tissue or an agent in vivo. A ligand canbe an antibody, an antibody fragment, an aptamer, a peptide, a vitamin,a carbohydrate, a protein or polypeptide, a receptor, e.g., cell-surfacereceptor, an adhesion molecule, a glycoprotein, a sugar residue, atherapeutic agent, a drug, a glycosaminoglycan, or any combinationthereof. For example, a ligand can be an antibody that recognizes acancer-cell specific antigen, rendering the cell capable ofpreferentially interacting with tumor cells to permit tumor-specificlocalization of a modified cell. A ligand can confer the ability of acell composition to accumulate in a tissue to be treated, since apreferred ligand may be capable of interacting with a target molecule onthe external face of a tissue to be treated. Ligands having limitedcross-reactivity to other tissues are generally preferred.

In some cases, a ligand can act as a homing moiety which permits thecell to target to a specific tissue or interact with a specific ligand.Such homing moieties can include, but are not limited to, any member ofa specific binding pair, antibodies, monoclonal antibodies, orderivatives or analogs thereof, including without limitation: Fvfragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2fragments, single domain antibodies, camelized antibodies and antibodyfragments, humanized antibodies and antibody fragments, and multivalentversions of the foregoing; multivalent binding reagents includingwithout limitation: monospecific or bispecific antibodies, such asdisulfide stabilized Fv fragments, scFv tandems ((SCFV)2 fragments),diabodies, tribodies or tetrabodies, which typically are covalentlylinked or otherwise stabilized (i.e., leucine zipper or helixstabilized) scFv fragments; and other homing moieties include forexample, aptamers, receptors, and fusion proteins.

In some embodiments, the homing moiety may be a surface-bound antibody,which can permit tuning of cell targeting specificity. This isespecially useful since highly specific antibodies can be raised againstan epitope of interest for the desired targeting site. In oneembodiment, multiple antibodies are expressed on the surface of a cell,and each antibody can have a different specificity for a desired target.Such approaches can increase the avidity and specificity of hominginteractions.

A skilled artisan can select any homing moiety based on the desiredlocalization or function of the cell, for example an estrogen receptorligand, such as tamoxifen, can target cells to estrogen-dependent breastcancer cells that have an increased number of estrogen receptors on thecell surface. Other non-limiting examples of ligand/receptorinteractions include CCRI (e.g., for treatment of inflamed joint tissuesor brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8(e.g., targeting to lymph node tissue), CCR6, CCR9, CCR10 (e.g., totarget to intestinal tissue), CCR4, CCR10 (e.g., for targeting to skin),CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., fortreatment of inflammation and inflammatory disorders, bone marrow),Alpha4beta7 (e.g., for intestinal mucosa targeting), VLA-4/VCAM-1 (e.g.,targeting to endothelium). In general, any receptor involved intargeting (e.g., cancer metastasis) can be harnessed for use in themethods and compositions described herein.

Modulation of Cell Lineage

Provided are methods of inducing an alteration in cell fate in a targetmammalian cell. The target mammalian cell may be a precursor cell andthe alteration may involve driving differentiation into a lineage, orblocking such differentiation. Alternatively, the target mammalian cellmay be a differentiated cell, and the cell fate alteration includesdriving de-differentiation into a pluripotent precursor cell, orblocking such de-differentiation, such as the dedifferentiation ofcancer cells into cancer stem cells. In situations where a change incell fate is desired, effective amounts of mRNAs encoding a cell fateinductive polypeptide is introduced into a target cell under conditionssuch that an alteration in cell fate is induced. In some embodiments,the modified mRNAs are useful to reprogram a subpopulation of cells froma first phenotype to a second phenotype. Such a reprogramming may betemporary or permanent.

Optionally, the reprogramming induces a target cell to adopt anintermediate phenotype.

Additionally, the methods of the present invention are particularlyuseful to generate induced pluripotent stem cells (iPS cells) because ofthe high efficiency of transfection, the ability to re-transfect cells,and the tenability of the amount of recombinant polypeptides produced inthe target cells. Further, the use of iPS cells generated using themethods described herein is expected to have a reduced incidence ofteratoma formation.

Also provided are methods of reducing cellular differentiation in atarget cell population. For example, a target cell population containingone or more precursor cell types is contacted with a composition havingan effective amount of a polynucleotides, primary constructs and mmRNAencoding a polypeptide, under conditions such that the polypeptide istranslated and reduces the differentiation of the precursor cell. Innon-limiting embodiments, the target cell population contains injuredtissue in a mammalian subject or tissue affected by a surgicalprocedure. The precursor cell is, e.g., a stromal precursor cell, aneural precursor cell, or a mesenchymal precursor cell.

In a specific embodiment, provided are polynucleotide, primary constructor mmRNA that encode one or more differentiation factors Gata4, Mef2cand Tbx4. These mRNA-generated factors are introduced into fibroblastsand drive the reprogramming into cardiomyocytes. Such a reprogrammingcan be performed in vivo, by contacting an mRNA-containing patch orother material to damaged cardiac tissue to facilitate cardiacregeneration. Such a process promotes cardiomyocyte genesis as opposedto fibrosis.

Mediation of Cell Death

In one embodiment, polynucleotides, primary constructs or mmRNAcompositions can be used to induce apoptosis in a cell (e.g., a cancercell) by increasing the expression of a death receptor, a death receptorligand or a combination thereof. This method can be used to induce celldeath in any desired cell and has particular usefulness in the treatmentof cancer where cells escape natural apoptotic signals.

Apoptosis can be induced by multiple independent signaling pathways thatconverge upon a final effector mechanism consisting of multipleinteractions between several “death receptors” and their ligands, whichbelong to the tumor necrosis factor (TNF) receptor/ligand superfamily.The best-characterized death receptors are CD95 (“Fas”), TNFRI (p55),death receptor 3 (DR3 or Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2). Thefinal effector mechanism of apoptosis may be the activation of a seriesof proteinases designated as caspases. The activation of these caspasesresults in the cleavage of a series of vital cellular proteins and celldeath. The molecular mechanism of death receptors/ligands-inducedapoptosis is well known in the art. For example, Fas/FasL-mediatedapoptosis is induced by binding of three FasL molecules which inducestrimerization of Fas receptor via C-terminus death domains (DDs), whichin turn recruits an adapter protein FADD (Fas-associated protein withdeath domain) and Caspase-8. The oligomerization of this trimolecularcomplex, Fas/FAIDD/caspase-8, results in proteolytic cleavage ofproenzyme caspase-8 into active caspase-8 that, in turn, initiates theapoptosis process by activating other downstream caspases throughproteolysis, including caspase-3. Death ligands in general are apoptoticwhen formed into trimers or higher order of structures. As monomers,they may serve as antiapoptotic agents by competing with the trimers forbinding to the death receptors.

In one embodiment, the oncology-related polynucleotides, primaryconstructs or mmRNA composition encodes for a death receptor (e.g., Fas,TRAIL, TRAMO, TNFR, TLR etc). Cells made to express a death receptor bytransfection of oncology-related polynucleotides, primary constructs andmmRNA become susceptible to death induced by the ligand that activatesthat receptor. Similarly, cells made to express a death ligand, e.g., ontheir surface, will induce death of cells with the receptor when thetransfected cell contacts the target cell. In another embodiment, theoncology-related polynucleotides, primary constructs and mmRNAcomposition encodes for a death receptor ligand (e.g., FasL, TNF, etc).In another embodiment, the oncology-related polynucleotides, primaryconstructs and mmRNA composition encodes a caspase (e.g., caspase 3,caspase 8, caspase 9 etc). Where cancer cells often exhibit a failure toproperly differentiate to a non-proliferative or controlledproliferative form, in another embodiment, the synthetic,polynucleotides, primary constructs and mmRNA composition encodes forboth a death receptor and its appropriate activating ligand. In anotherembodiment, the synthetic, polynucleotides, primary constructs and mmRNAcomposition encodes for a differentiation factor that when expressed inthe cancer cell, such as a cancer stem cell, will induce the cell todifferentiate to a non-pathogenic or nonself-renewing phenotype (e.g.,reduced cell growth rate, reduced cell division etc) or to induce thecell to enter a dormant cell phase (e.g., Go resting phase).

One of skill in the art will appreciate that the use ofapoptosis-inducing techniques may require that the oncology-relatedpolynucleotides, primary constructs or mmRNA are appropriately targetedto e.g., tumor cells to prevent unwanted wide-spread cell death. Thus,one can use a delivery mechanism (e.g., attached ligand or antibody,targeted liposome etc) that recognizes a cancer antigen such that theoncology-related polynucleotides, primary constructs or mmRNA areexpressed only in cancer cells.

VI. KITS AND DEVICES Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods of the present invention. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments.

In one aspect, the present invention provides kits comprising themolecules (oncology-related polynucleotides, oncology-related primaryconstructs or oncology-related mmRNA) of the invention. In oneembodiment, the kit comprises one or more functional antibodies orfunction fragments thereof.

Said kits can be for oncology-related protein production, comprising afirst oncology-related polynucleotide, oncology-related primaryconstruct or oncology-related mmRNA comprising a translatable region.The kit may further comprise packaging and instructions and/or adelivery agent to form a formulation composition. The delivery agent maycomprise a saline, a buffered solution, a lipidoid or any delivery agentdisclosed herein.

In one embodiment, the buffer solution may include sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution may include, but is not limited to, saline, saline with2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium. In a further embodiment, the buffersolutions may be precipitated or it may be lyophilized. The amount ofeach component may be varied to enable consistent, reproducible higherconcentration saline or simple buffer formulations. The components mayalso be varied in order to increase the stability of modified RNA in thebuffer solution over a period of time and/or under a variety ofconditions. In one aspect, the present invention provides kits foroncology-related protein production, comprising: an oncology-relatedpolynucleotide, oncology-related primary construct or oncology-relatedmmRNA comprising a translatable region, provided in an amount effectiveto produce a desired amount of an oncology-related protein encoded bythe translatable region when introduced into a target cell; a secondoncology-related polynucleotide comprising an inhibitory nucleic acid,provided in an amount effective to substantially inhibit the innateimmune response of the cell; and packaging and instructions.

In one aspect, the present invention provides kits for oncology-relatedprotein production, comprising an oncology-related polynucleotide,oncology-related primary construct or oncology-related mmRNA comprisinga translatable region, wherein the oncology-related polynucleotideexhibits reduced degradation by a cellular nuclease, and packaging andinstructions.

In one aspect, the present invention provides kits for oncology-relatedprotein production, comprising an oncology-related polynucleotide,oncology-related primary construct or oncology-related mmRNA comprisinga translatable region, wherein the oncology-related polynucleotideexhibits reduced degradation by a cellular nuclease, and a mammaliancell suitable for translation of the translatable region of the firstnucleic acid.

In one embodiment, the levels of Protein C may be measured byimmunoassay. The assay may be purchased and is available from any numberof suppliers including BioMerieux, Inc. (Durham, N.C.), AbbottLaboratories (Abbott Park, Ill.), Siemens Medical Solutions USA, Inc.(Malvern, Pa.), BIOPORTO® Diagnostics A/S (Gentofte, Denmark), USCN®Life Science Inc. (Houston, Tex.) or Roche Diagnostic Corporation(Indianapolis, Ind.). In this embodiment, the assay may be used toassess levels of Protein C or its activated form or a variant deliveredas or in response to administration of a modified mRNA molecule.

Devices

The present invention provides for devices which may incorporateoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA that encode oncology-related polypeptides ofinterest. These devices contain in a stable formulation the reagents tosynthesize an oncology-related polynucleotide in a formulation availableto be immediately delivered to a subject in need thereof, such as ahuman patient. Non-limiting examples of such a polypeptide of interestinclude a growth factor and/or angiogenesis stimulator for woundhealing, a peptide antibiotic to facilitate infection control, and anantigen to rapidly stimulate an immune response to a newly identifiedvirus.

Devices may also be used in conjunction with the present invention. Inone embodiment, a device is used to assess levels of a protein which hasbeen administered in the form of a modified mRNA. The device maycomprise a blood, urine or other biofluidic test. It may be as large asto include an automated central lab platform or a small decentralizedbench top device. It may be point of care or a handheld device. In thisembodiment, for example, Protein C or APC may be quatitated before,during or after treatment with a modified mRNA encoding Protein C (itszymogen), APC or any variants thereof. Protein C, also known asautoprothrombin IIA and blood coagulation factor XIV is a zymogen, orprecursor, of a serine protease which plays an important role in theregulation of blood coagulation and generation of fibrinolytic activityin vivo. It is synthesized in the liver as a single-chain polypeptidebut undergoes posttranslational processing to give rise to a two-chainintermediate. The intermediate form of Protein C is converted viathrombin-mediated cleavage of a 12-residue peptide from theamino-terminus of the heavy chain to of the molecule to the active form,known as “activated protein C” (APC). The device may be useful in drugdiscovery efforts as a companion diagnostic test associated with ProteinC, or APC treatment such as for sepsis or severe sepsis. In earlystudies it was suggested that APC had the ability to reduce mortality insevere sepsis. Following this line of work, clinical studies lead to theFDA approval of one compound, activated drotrecogin alfa (recombinantprotein C). However, in late 2011, the drug was withdrawn from sale inall markets following results of the PROWESS-SHOCK study, which showedthe study did not meet the primary endpoint of a statisticallysignificant reduction in 28-day all-cause mortality in patients withseptic shock. The present invention provides modified mRNA moleculeswhich may be used in the diagnosis and treatment of sepsis, severesepsis and septicemia which overcome prior issues or problems associatedwith increasing protein expression efficiencies in mammals.

In some embodiments the device is self-contained, and is optionallycapable of wireless remote access to obtain instructions for synthesisand/or analysis of the generated oncology-related polynucleotide,oncology-related primary construct or oncology-related mmRNA. The deviceis capable of mobile synthesis of at least one oncology-relatedpolynucleotide, oncology-related primary construct or oncology-relatedmmRNA and preferably an unlimited number of different oncology-relatedpolynucleotides, oncology-related primary constructs or oncology-relatedmmRNA. In certain embodiments, the device is capable of beingtransported by one or a small number of individuals. In otherembodiments, the device is scaled to fit on a benchtop or desk. In otherembodiments, the device is scaled to fit into a suitcase, backpack orsimilarly sized object. In another embodiment, the device may be a pointof care or handheld device. In further embodiments, the device is scaledto fit into a vehicle, such as a car, truck or ambulance, or a militaryvehicle such as a tank or personnel carrier. The information necessaryto generate a modified oncology-related mRNA encoding oncology-relatedpolypeptide of interest is present within a computer readable mediumpresent in the device.

In one embodiment, a device may be used to assess levels of anoncology-related protein which has been administered in the form of anoncology-related polynucleotide, oncology-related primary construct oroncology-related mmRNA. The device may comprise a blood, urine or otherbiofluidic test.

In some embodiments, the device is capable of communication (e.g.,wireless communication) with a database of nucleic acid and polypeptidesequences which may be oncology-related nucleic acid andoncology-related polypeptide sequences. The device contains at least onesample block for insertion of one or more sample vessels. Such samplevessels are capable of accepting in liquid or other form any number ofmaterials such as template DNA, nucleotides, enzymes, buffers, and otherreagents. The sample vessels are also capable of being heated and cooledby contact with the sample block. The sample block is generally incommunication with a device base with one or more electronic controlunits for the at least one sample block. The sample block preferablycontains a heating module, such heating molecule capable of heatingand/or cooling the sample vessels and contents thereof to temperaturesbetween about −20 C and above +100 C. The device base is incommunication with a voltage supply such as a battery or externalvoltage supply. The device also contains means for storing anddistributing the materials for RNA synthesis.

Optionally, the sample block contains a module for separating thesynthesized nucleic acids. Alternatively, the device contains aseparation module operably linked to the sample block. Preferably thedevice contains a means for analysis of the synthesized nucleic acid.Such analysis includes sequence identity (demonstrated such as byhybridization), absence of non-desired sequences, measurement ofintegrity of synthesized mRNA (such has by microfluidic viscometrycombined with spectrophotometry), and concentration and/or potency ofmodified RNA (such as by spectrophotometry).

In certain embodiments, the device is combined with a means fordetection of pathogens present in a biological material obtained from asubject, e.g., the IBIS PLEX-ID system (Abbott, Abbott Park, Ill.) formicrobial identification.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662; each of which is hereinincorporated by reference in their entirety. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 (herein incorporated by reference in itsentirety) and functional equivalents thereof. Jet injection deviceswhich deliver liquid compositions to the dermis via a liquid jetinjector and/or via a needle which pierces the stratum corneum andproduces a jet which reaches the dermis are suitable. Jet injectiondevices are described, for example, in U.S. Pat. Nos. 5,480,381;5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911;5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627;5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; andPCT publications WO 97/37705 and WO 97/13537; each of which are whereinincorporated by reference in their entirety. Ballistic powder/particledelivery devices which use compressed gas to accelerate vaccine inpowder form through the outer layers of the skin to the dermis aresuitable. Alternatively or additionally, conventional syringes may beused in the classical mantoux method of intradermal administration.

In some embodiments, the device may be a pump or comprise a catheter foradministration of compounds or compositions of the invention across theblood brain barrier. Such devices include but are not limited to apressurized olfactory delivery device, iontophoresis devices,multi-layered microfluidic devices, and the like. Such devices may beportable or stationary. They may be implantable or externally tetheredto the body or combinations thereof.

Devices for administration may be employed to deliver theoncology-related polynucleotides, oncology-related primary constructs oroncology-related mmRNA of the present invention according to single,multi- or split-dosing regimens taught herein. Such devices aredescribed below.

Method and devices known in the art for multi-administration to cells,organs and tissues are contemplated for use in conjunction with themethods and compositions disclosed herein as embodiments of the presentinvention. These include, for example, those methods and devices havingmultiple needles, hybrid devices employing for example lumens orcatheters as well as devices utilizing heat, electric current orradiation driven mechanisms.

According to the present invention, these multi-administration devicesmay be utilized to deliver the single, multi- or split dosescontemplated herein.

A method for delivering therapeutic agents to a solid tissue has beendescribed by Bahrami et al. and is taught for example in US PatentPublication 20110230839, the contents of which are incorporated hereinby reference in their entirety. According to Bahrami, an array ofneedles is incorporated into a device which delivers a substantiallyequal amount of fluid at any location in said solid tissue along eachneedle's length.

A device for delivery of biological material across the biologicaltissue has been described by Kodgule et al. and is taught for example inUS Patent Publication 20110172610, the contents of which areincorporated herein by reference in their entirety. According toKodgule, multiple hollow micro-needles made of one or more metals andhaving outer diameters from about 200 microns to about 350 microns andlengths of at least 100 microns are incorporated into the device whichdelivers peptides, proteins, carbohydrates, nucleic acid molecules,lipids and other pharmaceutically active ingredients or combinationsthereof.

A delivery probe for delivering a therapeutic agent to a tissue has beendescribed by Gunday et al. and is taught for example in US PatentPublication 20110270184, the contents of each of which are incorporatedherein by reference in their entirety. According to Gunday, multipleneedles are incorporated into the device which moves the attachedcapsules between an activated position and an inactivated position toforce the agent out of the capsules through the needles.

A multiple-injection medical apparatus has been described by Assaf andis taught for example in US Patent Publication 20110218497, the contentsof which are incorporated herein by reference in their entirety.According to Assaf, multiple needles are incorporated into the devicewhich has a chamber connected to one or more of said needles and a meansfor continuously refilling the chamber with the medical fluid after eachinjection.

In one embodiment, the oncology-related polynucleotide, oncology-relatedprimary construct, or oncology-related mmRNA is administeredsubcutaneously or intramuscularly via at least 3 needles to threedifferent, optionally adjacent, sites simultaneously, or within a 60minutes period (e.g., administration to 4, 5, 6, 7, 8, 9, or 10 sitessimultaneously or within a 60 minute period). The split doses can beadministered simultaneously to adjacent tissue using the devicesdescribed in U.S. Patent Publication Nos. 20110230839 and 20110218497,each of which is incorporated herein by reference in their entirety.

An at least partially implantable system for injecting a substance intoa patient's body, in particular a penis erection stimulation system hasbeen described by Forsell and is taught for example in US PatentPublication 20110196198, the contents of which are incorporated hereinby reference in their entirety. According to Forsell, multiple needlesare incorporated into the device which is implanted along with one ormore housings adjacent the patient's left and right corpora cavernosa. Areservoir and a pump are also implanted to supply drugs through theneedles.

A method for the transdermal delivery of a therapeutic effective amountof iron has been described by Berenson and is taught for example in USPatent Publication 20100130910, the contents of which are incorporatedherein by reference in their entirety. According to Berenson, multipleneedles may be used to create multiple micro channels in stratum corneumto enhance transdermal delivery of the ionic iron on an iontophoreticpatch.

A method for delivery of biological material across the biologicaltissue has been described by Kodgule et al and is taught for example inUS Patent Publication 20110196308, the contents of which areincorporated herein by reference in their entirety. According toKodgule, multiple biodegradable microneedles containing a therapeuticactive ingredient are incorporated in a device which delivers proteins,carbohydrates, nucleic acid molecules, lipids and other pharmaceuticallyactive ingredients or combinations thereof.

A transdermal patch comprising a botulinum toxin composition has beendescribed by Donovan and is taught for example in US Patent Publication20080220020, the contents of which are incorporated herein by referencein their entirety. According to Donovan, multiple needles areincorporated into the patch which delivers botulinum toxin under stratumcorneum through said needles which project through the stratum corneumof the skin without rupturing a blood vessel.

A small, disposable drug reservoir, or patch pump, which can holdapproximately 0.2 to 15 mL of liquid formulations can be placed on theskin and deliver the formulation continuously subcutaneously using asmall bore needed (e.g., 26 to 34 gauge). As non-limiting examples, thepatch pump may be 50 mm by 76 mm by 20 mm spring loaded having a 30 to34 gauge needle (BD™ Microinfuser, Franklin Lakes N.J.), 41 mm by 62 mmby 17 mm with a 2 mL reservoir used for drug delivery such as insulin(OMNIPOD®, Insulet Corporation Bedford, Mass.), or 43-60 mm diameter, 10mm thick with a 0.5 to 10 mL reservoir (PATCHPUMP®, SteadyMedTherapeutics, San Francisco, Calif.). Further, the patch pump may bebattery powered and/or rechargeable.

A cryoprobe for administration of an active agent to a location ofcryogenic treatment has been described by Toubia and is taught forexample in US Patent Publication 20080140061, the contents of which areincorporated herein by reference in their entirety. According to Toubia,multiple needles are incorporated into the probe which receives theactive agent into a chamber and administers the agent to the tissue.

A method for treating or preventing inflammation or promoting healthyjoints has been described by Stock et al and is taught for example in USPatent Publication 20090155186, the contents of which are incorporatedherein by reference in their entirety. According to Stock, multipleneedles are incorporated in a device which administers compositionscontaining signal transduction modulator compounds.

A multi-site injection system has been described by Kimmell et al. andis taught for example in US Patent Publication 20100256594, the contentsof which are incorporated herein by reference in their entirety.According to Kimmell, multiple needles are incorporated into a devicewhich delivers a medication into a stratum corneum through the needles.

A method for delivering interferons to the intradermal compartment hasbeen described by Dekker et al. and is taught for example in US PatentPublication 20050181033, the contents of which are incorporated hereinby reference in their entirety. According to Dekker, multiple needleshaving an outlet with an exposed height between 0 and 1 mm areincorporated into a device which improves pharmacokinetics andbioavailability by delivering the substance at a depth between 0.3 mmand 2 mm.

A method for delivering genes, enzymes and biological agents to tissuecells has described by Desai and is taught for example in US PatentPublication 20030073908, the contents of which are incorporated hereinby reference in their entirety. According to Desai, multiple needles areincorporated into a device which is inserted into a body and delivers amedication fluid through said needles.

A method for treating cardiac arrhythmias with fibroblast cells has beendescribed by Lee et al and is taught for example in US PatentPublication 20040005295, the contents of which are incorporated hereinby reference in their entirety. According to Lee, multiple needles areincorporated into the device which delivers fibroblast cells into thelocal region of the tissue.

A method using a magnetically controlled pump for treating a brain tumorhas been described by Shachar et al. and is taught for example in U.S.Pat. No. 7,799,012 (method) and 7799016 (device), the contents of whichare incorporated herein by reference in their entirety. AccordingShachar, multiple needles were incorporated into the pump which pushes amedicating agent through the needles at a controlled rate.

Methods of treating functional disorders of the bladder in mammalianfemales have been described by Versi et al. and are taught for examplein U.S. Pat. No. 8,029,496, the contents of which are incorporatedherein by reference in their entirety. According to Versi, an array ofmicro-needles is incorporated into a device which delivers a therapeuticagent through the needles directly into the trigone of the bladder.

A micro-needle transdermal transport device has been described by Angelet al and is taught for example in U.S. Pat. No. 7,364,568, the contentsof which are incorporated herein by reference in their entirety.According to Angel, multiple needles are incorporated into the devicewhich transports a substance into a body surface through the needleswhich are inserted into the surface from different directions. Themicro-needle transdermal transport device may be a solid micro-needlesystem or a hollow micro-needle system. As a non-limiting example, thesolid micro-needle system may have up to a 0.5 mg capacity, with300-1500 solid micro-needles per cm² about 150-700 μm tall coated with adrug. The micro-needles penetrate the stratum corneum and remain in theskin for short duration (e.g., 20 seconds to 15 minutes). In anotherexample, the hollow micro-needle system has up to a 3 mL capacity todeliver liquid formulations using 15-20 microneedles per cm2 beingapproximately 950 μm tall. The micro-needles penetrate the skin to allowthe liquid formulations to flow from the device into the skin. Thehollow micro-needle system may be worn from 1 to 30 minutes depending onthe formulation volume and viscocity.

A device for subcutaneous infusion has been described by Dalton et aland is taught for example in U.S. Pat. No. 7,150,726, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Dalton, multiple needles are incorporated into the device whichdelivers fluid through the needles into a subcutaneous tissue.

A device and a method for intradermal delivery of vaccines and genetherapeutic agents through microcannula have been described by Miksztaet al. and are taught for example in U.S. Pat. No. 7,473,247, thecontents of which are incorporated herein by reference in theirentirety. According to Mitszta, at least one hollow micro-needle isincorporated into the device which delivers the vaccines to thesubject's skin to a depth of between 0.025 mm and 2 mm.

A method of delivering insulin has been described by Pettis et al and istaught for example in U.S. Pat. No. 7,722,595, the contents of which areincorporated herein by reference in their entirety. According to Pettis,two needles are incorporated into a device wherein both needles insertessentially simultaneously into the skin with the first at a depth ofless than 2.5 mm to deliver insulin to intradermal compartment and thesecond at a depth of greater than 2.5 mm and less than 5.0 mm to deliverinsulin to subcutaneous compartment.

Cutaneous injection delivery under suction has been described byKochamba et al. and is taught for example in U.S. Pat. No. 6,896,666,the contents of which are incorporated herein by reference in theirentirety. According to Kochamba, multiple needles in relative adjacencywith each other are incorporated into a device which injects a fluidbelow the cutaneous layer.

A device for withdrawing or delivering a substance through the skin hasbeen described by Down et al and is taught for example in U.S. Pat. No.6,607,513, the contents of which are incorporated herein by reference intheir entirety. According to Down, multiple skin penetrating memberswhich are incorporated into the device have lengths of about 100 micronsto about 2000 microns and are about 30 to 50 gauge.

A device for delivering a substance to the skin has been described byPalmer et al and is taught for example in U.S. Pat. No. 6,537,242, thecontents of which are incorporated herein by reference in theirentirety. According to Palmer, an array of micro-needles is incorporatedinto the device which uses a stretching assembly to enhance the contactof the needles with the skin and provides a more uniform delivery of thesubstance.

A perfusion device for localized drug delivery has been described byZamoyski and is taught for example in U.S. Pat. No. 6,468,247, thecontents of which are incorporated herein by reference in theirentirety. According to Zamoyski, multiple hypodermic needles areincorporated into the device which injects the contents of thehypodermics into a tissue as said hypodermics are being retracted.

A method for enhanced transport of drugs and biological molecules acrosstissue by improving the interaction between micro-needles and human skinhas been described by Prausnitz et al. and is taught for example in U.S.Pat. No. 6,743,211, the contents of which are incorporated herein byreference in their entirety. According to Prausnitz, multiplemicro-needles are incorporated into a device which is able to present amore rigid and less deformable surface to which the micro-needles areapplied.

A device for intraorgan administration of medicinal agents has beendescribed by Ting et al and is taught for example in U.S. Pat. No.6,077,251, the contents of which are incorporated herein by reference intheir entirety. According to Ting, multiple needles having side openingsfor enhanced administration are incorporated into a device which byextending and retracting said needles from and into the needle chamberforces a medicinal agent from a reservoir into said needles and injectssaid medicinal agent into a target organ.

A multiple needle holder and a subcutaneous multiple channel infusionport has been described by Brown and is taught for example in U.S. Pat.No. 4,695,273, the contents of which are incorporated herein byreference in their entirety. According to Brown, multiple needles on theneedle holder are inserted through the septum of the infusion port andcommunicate with isolated chambers in said infusion port.

A dual hypodermic syringe has been described by Horn and is taught forexample in U.S. Pat. No. 3,552,394, the contents of which areincorporated herein by reference in their entirety. According to Horn,two needles incorporated into the device are spaced apart less than 68mm and may be of different styles and lengths, thus enabling injectionsto be made to different depths.

A syringe with multiple needles and multiple fluid compartments has beendescribed by Hershberg and is taught for example in U.S. Pat. No.3,572,336, the contents of which are incorporated herein by reference intheir entirety. According to Hershberg, multiple needles areincorporated into the syringe which has multiple fluid compartments andis capable of simultaneously administering incompatible drugs which arenot able to be mixed for one injection.

A surgical instrument for intradermal injection of fluids has beendescribed by Eliscu et al. and is taught for example in U.S. Pat. No.2,588,623, the contents of which are incorporated herein by reference intheir entirety. According to Eliscu, multiple needles are incorporatedinto the instrument which injects fluids intradermally with a widerdisperse.

An apparatus for simultaneous delivery of a substance to multiple breastmilk ducts has been described by Hung and is taught for example in EP1818017, the contents of which are incorporated herein by reference intheir entirety. According to Hung, multiple lumens are incorporated intothe device which inserts though the orifices of the ductal networks anddelivers a fluid to the ductal networks.

A catheter for introduction of medications to the tissue of a heart orother organs has been described by Tkebuchava and is taught for examplein WO2006138109, the contents of which are incorporated herein byreference in their entirety. According to Tkebuchava, two curved needlesare incorporated which enter the organ wall in a flattened trajectory.

Devices for delivering medical agents have been described by Mckay etal. and are taught for example in WO2006118804, the content of which areincorporated herein by reference in their entirety. According to Mckay,multiple needles with multiple orifices on each needle are incorporatedinto the devices to facilitate regional delivery to a tissue, such asthe interior disc space of a spinal disc.

A method for directly delivering an immunomodulatory substance into anintradermal space within a mammalian skin has been described by Pettisand is taught for example in WO2004020014, the contents of which areincorporated herein by reference in their entirety. According to Pettis,multiple needles are incorporated into a device which delivers thesubstance through the needles to a depth between 0.3 mm and 2 mm.

Methods and devices for administration of substances into at least twocompartments in skin for systemic absorption and improvedpharmacokinetics have been described by Pettis et al. and are taught forexample in WO2003094995, the contents of which are incorporated hereinby reference in their entirety. According to Pettis, multiple needleshaving lengths between about 300 μm and about 5 mm are incorporated intoa device which delivers to intradermal and subcutaneous tissuecompartments simultaneously.

A drug delivery device with needles and a roller has been described byZimmerman et al. and is taught for example in WO2012006259, the contentsof which are incorporated herein by reference in their entirety.According to Zimmerman, multiple hollow needles positioned in a rollerare incorporated into the device which delivers the content in areservoir through the needles as the roller rotates.

A drug delivery device such as a stent is known in the art and is taughtfor example in U.S. Pat. No. 8,333,799, U.S. Pub. Nos. US20060020329,US20040172127 and US20100161032; the contents of each of which areherein incorporated by reference in their entirety. Formulations of thepolynucleotides, primary constructs, mmRNA described herein may bedelivered using stents. Additionally, stents used herein may be able todeliver multiple polynucleotides, primary constructs and/or mmRNA and/orformulations at the same or varied rates of delivery. Non-limitingexamples of manufacturers of stents include CORDIS® (Miami, Fla.)(CYPHER®), Boston Scientific Corporation (Natick, Mass.) (TAXUS®),Medtronic (Minneapolis, Minn.) (ENDEAVOUR®) and Abbott (Abbott Park,Ill.) (XIENCE V®).

Methods and Devices Utilizing Catheters and/or Lumens

Methods and devices using catheters and lumens may be employed toadminister the oncology-related mmRNA of the present invention on asingle, multi- or split dosing schedule. Such methods and devices aredescribed below.

A catheter-based delivery of skeletal myoblasts to the myocardium ofdamaged hearts has been described by Jacoby et al and is taught forexample in US Patent Publication 20060263338, the contents of which areincorporated herein by reference in their entirety. According to Jacoby,multiple needles are incorporated into the device at least part of whichis inserted into a blood vessel and delivers the cell compositionthrough the needles into the localized region of the subject's heart.

An apparatus for treating asthma using neurotoxin has been described byDeem et al and is taught for example in US Patent Publication20060225742, the contents of which are incorporated herein by referencein their entirety. According to Deem, multiple needles are incorporatedinto the device which delivers neurotoxin through the needles into thebronchial tissue.

A method for administering multiple-component therapies has beendescribed by Nayak and is taught for example in U.S. Pat. No. 7,699,803,the contents of which are incorporated herein by reference in theirentirety. According to Nayak, multiple injection cannulas may beincorporated into a device wherein depth slots may be included forcontrolling the depth at which the therapeutic substance is deliveredwithin the tissue.

A surgical device for ablating a channel and delivering at least onetherapeutic agent into a desired region of the tissue has been describedby McIntyre et al and is taught for example in U.S. Pat. No. 8,012,096,the contents of which are incorporated herein by reference in theirentirety. According to McIntyre, multiple needles are incorporated intothe device which dispenses a therapeutic agent into a region of tissuesurrounding the channel and is particularly well suited fortransmyocardial revascularization operations.

Methods of treating functional disorders of the bladder in mammalianfemales have been described by Versi et al and are taught for example inU.S. Pat. No. 8,029,496, the contents of which are incorporated hereinby reference in their entirety. According to Versi, an array ofmicro-needles is incorporated into a device which delivers a therapeuticagent through the needles directly into the trigone of the bladder.

A device and a method for delivering fluid into a flexible biologicalbarrier have been described by Yeshurun et al. and are taught forexample in U.S. Pat. No. 7,998,119 (device) and U.S. Pat. No. 8,007,466(method), the contents of which are incorporated herein by reference intheir entirety. According to Yeshurun, the micro-needles on the devicepenetrate and extend into the flexible biological barrier and fluid isinjected through the bore of the hollow micro-needles.

A method for epicardially injecting a substance into an area of tissueof a heart having an epicardial surface and disposed within a torso hasbeen described by Bonner et al and is taught for example in U.S. Pat.No. 7,628,780, the contents of which are incorporated herein byreference in their entirety. According to Bonner, the devices haveelongate shafts and distal injection heads for driving needles intotissue and injecting medical agents into the tissue through the needles.

A device for sealing a puncture has been described by Nielsen et al andis taught for example in U.S. Pat. No. 7,972,358, the contents of whichare incorporated herein by reference in their entirety. According toNielsen, multiple needles are incorporated into the device whichdelivers a closure agent into the tissue surrounding the puncture tract.

A method for myogenesis and angiogenesis has been described by Chiu etal. and is taught for example in U.S. Pat. No. 6,551,338, the contentsof which are incorporated herein by reference in their entirety.According to Chiu, 5 to 15 needles having a maximum diameter of at least1.25 mm and a length effective to provide a puncture depth of 6 to 20 mmare incorporated into a device which inserts into proximity with amyocardium and supplies an exogeneous angiogenic or myogenic factor tosaid myocardium through the conduits which are in at least some of saidneedles.

A method for the treatment of prostate tissue has been described byBolmsj et al. and is taught for example in U.S. Pat. No. 6,524,270, thecontents of which are incorporated herein by reference in theirentirety. According to Bolmsj, a device comprising a catheter which isinserted through the urethra has at least one hollow tip extendible intothe surrounding prostate tissue. An astringent and analgesic medicine isadministered through said tip into said prostate tissue.

A method for infusing fluids to an intraosseous site has been describedby Findlay et al. and is taught for example in U.S. Pat. No. 6,761,726,the contents of which are incorporated herein by reference in theirentirety. According to Findlay, multiple needles are incorporated into adevice which is capable of penetrating a hard shell of material coveredby a layer of soft material and delivers a fluid at a predetermineddistance below said hard shell of material.

A device for injecting medications into a vessel wall has been describedby Vigil et al. and is taught for example in U.S. Pat. No. 5,713,863,the contents of which are incorporated herein by reference in theirentirety. According to Vigil, multiple injectors are mounted on each ofthe flexible tubes in the device which introduces a medication fluidthrough a multi-lumen catheter, into said flexible tubes and out of saidinjectors for infusion into the vessel wall.

A catheter for delivering therapeutic and/or diagnostic agents to thetissue surrounding a bodily passageway has been described by Faxon etal. and is taught for example in U.S. Pat. No. 5,464,395, the contentsof which are incorporated herein by reference in their entirety.According to Faxon, at least one needle cannula is incorporated into thecatheter which delivers the desired agents to the tissue through saidneedles which project outboard of the catheter.

Balloon catheters for delivering therapeutic agents have been describedby Orr and are taught for example in WO2010024871, the contents of whichare incorporated herein by reference in their entirety. According toOrr, multiple needles are incorporated into the devices which deliverthe therapeutic agents to different depths within the tissue. In anotheraspect, drug-eluting balloons may be used to deliver the formulationsdescribed herein. The drug-eluting balloons may be used in target lesionapplications such as, but are not limited to, in-stent restenosis,treating lesion in tortuous vessels, bifurcation lesions,femoral/popliteal lesions and below the knee lesions.

A device for delivering therapeutic agents (e.g., oncology-relatedpolynucleotides, primary constructs or mmRNA) to tissue disposed about alumin has been described by Perry et al. and is taught for example inU.S. Pat. Pub. US20100125239, the contents of which are hereinincorporated by reference in their entirety. According to Perry, thecatheter has a balloon which may be coated with a therapeutic agent bymethods known in the art and described in Perry. When the balloonexpands, the therapeutic agent will contact the surrounding tissue. Thedevice may additionally have a heat source to change the temperature ofthe coating on the balloon to release the therapeutic agent to thetissue.

Methods and Devices Utilizing Electrical Current

Methods and devices utilizing electric current may be employed todeliver the mmRNA of the present invention according to the single,multi- or split dosing regimens taught herein. Such methods and devicesare described below.

An electro collagen induction therapy device has been described byMarquez and is taught for example in US Patent Publication 20090137945,the contents of which are incorporated herein by reference in theirentirety. According to Marquez, multiple needles are incorporated intothe device which repeatedly pierce the skin and draw in the skin aportion of the substance which is applied to the skin first.

An electrokinetic system has been described by Etheredge et al. and istaught for example in US Patent Publication 20070185432, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Etheredge, micro-needles are incorporated into a device which drivesby an electrical current the medication through the needles into thetargeted treatment site.

An iontophoresis device has been described by Matsumura et al. and istaught for example in U.S. Pat. No. 7,437,189, the contents of which areincorporated herein by reference in their entirety. According toMatsumura, multiple needles are incorporated into the device which iscapable of delivering ionizable drug into a living body at higher speedor with higher efficiency.

Intradermal delivery of biologically active agents by needle-freeinjection and electroporation has been described by Hoffmann et al andis taught for example in U.S. Pat. No. 7,171,264, the contents of whichare incorporated herein by reference in their entirety. According toHoffmann, one or more needle-free injectors are incorporated into anelectroporation device and the combination of needle-free injection andelectroporation is sufficient to introduce the agent into cells in skin,muscle or mucosa.

A method for electropermeabilization-mediated intracellular delivery hasbeen described by Lundkvist et al. and is taught for example in U.S.Pat. No. 6,625,486, the contents of which are incorporated herein byreference in their entirety. According to Lundkvist, a pair of needleelectrodes is incorporated into a catheter. Said catheter is positionedinto a body lumen followed by extending said needle electrodes topenetrate into the tissue surrounding said lumen. Then the deviceintroduces an agent through at least one of said needle electrodes andapplies electric field by said pair of needle electrodes to allow saidagent pass through the cell membranes into the cells at the treatmentsite.

A delivery system for transdermal immunization has been described byLevin et al. and is taught for example in WO2006003659, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Levin, multiple electrodes are incorporated into the device whichapplies electrical energy between the electrodes to generate microchannels in the skin to facilitate transdermal delivery.

A method for delivering RF energy into skin has been described bySchomacker and is taught for example in WO2011163264, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Schomacker, multiple needles are incorporated into a device whichapplies vacuum to draw skin into contact with a plate so that needlesinsert into skin through the holes on the plate and deliver RF energy.

VII. DEFINITIONS

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl.

About: As used herein, the term “about” means +/−10% of the recitedvalue.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there may be an overlap of an effect of each agent on thepatient. In some embodiments, they are administered within about 60, 30,15, 10, 5, or 1 minute of one another. In some embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Antigens of interest or desired antigens: As used herein, the terms“antigens of interest” or “desired antigens” include those proteins andother biomolecules provided herein that are immunospecifically bound bythe antibodies and fragments, mutants, variants, and alterations thereofdescribed herein. Examples of antigens of interest include, but are notlimited to, insulin, insulin-like growth factor, hGH, tPA, cytokines,such as interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega orIFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF beta,TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety which is capable of or maintains at leasttwo functions. The functions may effect the same outcome or a differentoutcome. The structure that produces the function may be the same ordifferent. For example, bifunctional modified RNAs of the presentinvention may encode a cytotoxic peptide (a first function) while thosenucleosides which comprise the encoding RNA are, in and of themselves,cytotoxic (second function). In this example, delivery of thebifunctional modified RNA to a cancer cell would produce not only apeptide or protein molecule which may ameliorate or treat the cancer butwould also deliver a cytotoxic payload of nucleosides to the cell shoulddegradation, instead of translation of the modified RNA, occur.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, anoncology-related polynucleotide, oncology-related primary construct oroncology-related mmRNA of the present invention may be consideredbiologically active if even a portion of the oncology-relatedpolynucleotide, oncology-related primary construct or oncology-relatedmmRNA is biologically active or mimics an activity consideredbiologically relevant.

Cancer: As used herein, the term “cancer” in a subject refers to thepresence of cells possessing characteristics, such as uncontrolledproliferation, immortality, metastatic potential, rapid growth andproliferation rate, and certain characteristic morphological features.Often, cancer cells will be in the form of a tumor, but such cells mayexist alone within a subject, or may circulate in the blood stream asindependent cells, such as leukemic cells.

Cell growth: As used herein, the term “cell growth” is principallyassociated with growth in cell numbers, which occurs by means of cellreproduction (i.e. proliferation) when the rate of the latter is greaterthan the rate of cell death (e.g. by apoptosis or necrosis).

Chemical terms: The following provides the definition of variouschemical terms from “acyl” to “thiol.”

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,propionyl, butanoyl and the like. Exemplary unsubstituted acyl groupsinclude from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In someembodiments, the alkyl group is further substituted with 1, 2, 3, or 4substituents as described herein.

The term “acylamino,” as used herein, represents an acyl group, asdefined herein, attached to the parent molecular group though an aminogroup, as defined herein (i.e., —N(R^(N1))—C(O)—R, where R is H or anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group and R^(N1) isas defined herein). Exemplary unsubstituted acylamino groups includefrom 1 to 41 carbons (e.g., from 1 to 7, from 1 to 13, from 1 to 21,from 2 to 7, from 2 to 13, from 2 to 21, or from 2 to 41 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “acyloxy,” as used herein, represents an acyl group, as definedherein, attached to the parent molecular group though an oxygen atom(i.e., —O—C(O)—R, where R is H or an optionally substituted C₁₋₆, C₁₋₁₀,or C₁₋₂₀ alkyl group). Exemplary unsubstituted acyloxy groups includefrom 1 to 21 carbons (e.g., from 1 to 7 or from 1 to 11 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “alkaryl,” as used herein, represents an aryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkaryl groups arefrom 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, suchas C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀ alk-C₆₋₁₀ aryl).In some embodiments, the alkylene and the aryl each can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein forthe respective groups. Other groups preceded by the prefix “alk-” aredefined in the same manner, where “alk” refers to a C₁₋₆ alkylene,unless otherwise noted, and the attached chemical structure is asdefined herein.

The term “alkcycloalkyl” represents a cycloalkyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein (e.g., an alkylene group of from 1 to 4, from 1to 6, from 1 to 10, or form 1 to 20 carbons). In some embodiments, thealkylene and the cycloalkyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, andthe like. Alkenyls include both cis and trans isomers. Alkenyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from amino, aryl, cycloalkyl, orheterocyclyl (e.g., heteroaryl), as defined herein, or any of theexemplary alkyl substituent groups described herein.

The term “alkenyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkenyl group (e.g., C₂₋₆ or C₂₋₁₀ alkenyl), unlessotherwise specified. Exemplary alkenyloxy groups include ethenyloxy,propenyloxy, and the like. In some embodiments, the alkenyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “alkheteroaryl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheteroaryl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂heteroaryl, or C₂₋₂₀ alk-C₁₋₁₂ heteroaryl). In some embodiments, thealkylene and the heteroaryl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.Alkheteroaryl groups are a subset of alkheterocyclyl groups.

The term “alkheterocyclyl” represents a heterocyclyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheterocyclyl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heterocyclyl, C₁₋₁₀ alk-C₁₋₁₂heterocyclyl, or C₁₋₂₀ alk-C₁₋₁₂ heterocyclyl). In some embodiments, thealkylene and the heterocyclyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkoxy” represents a chemical substituent of formula —OR,where R is a C₁₋₂₀ alkyl group (e.g., C₁₋₆ or C₁₋₁₀ alkyl), unlessotherwise specified. Exemplary alkoxy groups include methoxy, ethoxy,propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. Insome embodiments, the alkyl group can be further substituted with 1, 2,3, or 4 substituent groups as defined herein (e.g., hydroxy or alkoxy).

The term “alkoxyalkoxy” represents an alkoxy group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkoxy groupsinclude between 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20carbons, such as C₁₋₆ alkoxy-C₁₋₆ alkoxy, C₁₋₁₀ alkoxy-C₁₋₁₀ alkoxy, orC₁₋₂₀ alkoxy-C₁₋₂₀ alkoxy). In some embodiments, the each alkoxy groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkoxyalkyl” represents an alkyl group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups includebetween 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20 carbons,such as C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₁₀ alkoxy-C₁₋₁₀ alkyl, or C₁₋₂₀alkoxy-C₁₋₂₀ alkyl). In some embodiments, the alkyl and the alkoxy eachcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective group.

The term “alkoxycarbonyl,” as used herein, represents an alkoxy, asdefined herein, attached to the parent molecular group through acarbonyl atom (e.g., —C(O)—OR, where R is H or an optionally substitutedC₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplary unsubstitutedalkoxycarbonyl include from 1 to 21 carbons (e.g., from 1 to 11 or from1 to 7 carbons). In some embodiments, the alkoxy group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The term “alkoxycarbonylalkoxy,” as used herein, represents an alkoxygroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., —O-alkyl-C(O)—OR, where R is anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplaryunsubstituted alkoxycarbonylalkoxy include from 3 to 41 carbons (e.g.,from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ alkoxy, C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ alkoxy, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkoxy). Insome embodiments, each alkoxy group is further independently substitutedwith 1, 2, 3, or 4 substituents, as described herein (e.g., a hydroxygroup).

The term “alkoxycarbonylalkyl,” as used herein, represents an alkylgroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., -alkyl-C(O)—OR, where R is an optionallysubstituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group). Exemplary unsubstitutedalkoxycarbonylalkyl include from 3 to 41 carbons (e.g., from 3 to 10,from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31 carbons, suchas C₁₋₆ alkoxycarbonyl-C₁₋₆ alkyl, C₁₋₁₀ alkoxycarbonyl-C₁₋₁₀ alkyl, orC₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkyl). In some embodiments, each alkyl andalkoxy group is further independently substituted with 1, 2, 3, or 4substituents as described herein (e.g., a hydroxy group).

The term “alkyl,” as used herein, is inclusive of both straight chainand branched chain saturated groups from 1 to 20 carbons (e.g., from 1to 10 or from 1 to 6), unless otherwise specified. Alkyl groups areexemplified by methyl, ethyl, n- and isopropyl, n-, sec-, iso- andtert-butyl, neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as definedherein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino(i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8)hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein. For example, the alkylene group of aC₁-alkaryl can be further substituted with an oxo group to afford therespective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term“C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylenegroups having between x and y carbons. Exemplary values for x are 1, 2,3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, orC₂₋₂₀ alkylene). In some embodiments, the alkylene can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein foran alkyl group.

The term “alkylsulfinyl,” as used herein, represents an alkyl groupattached to the parent molecular group through an —S(O)— group.Exemplary unsubstituted alkylsulfinyl groups are from 1 to 6, from 1 to10, or from 1 to 20 carbons. In some embodiments, the alkyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein.

The term “alkylsulfinylalkyl,” as used herein, represents an alkylgroup, as defined herein, substituted by an alkylsulfinyl group.Exemplary unsubstituted alkylsulfinylalkyl groups are from 2 to 12, from2 to 20, or from 2 to 40 carbons. In some embodiments, each alkyl groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

The term “alkynyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkynyl group (e.g., C₂₋₆ or C₂₋₁₀ alkynyl), unlessotherwise specified. Exemplary alkynyloxy groups include ethynyloxy,propynyloxy, and the like. In some embodiments, the alkynyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “amidine,” as used herein, represents a —C(═NH)NH₂ group.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl, sulfoalkyl,heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g.,alkheteroaryl), wherein each of these recited R^(N1) groups can beoptionally substituted, as defined herein for each group; or two R^(N1)combine to form a heterocyclyl or an N-protecting group, and whereineach R^(N2) is, independently, H, alkyl, or aryl. The amino groups ofthe invention can be an unsubstituted amino (i.e., —NH₂) or asubstituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, aminois —NH₂ or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2)2, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl,sulfoalkyl, or aryl, and each R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). In some embodiments, the amino acid isattached to the parent molecular group by a carbonyl group, where theside chain or amino group is attached to the carbonyl group. Exemplaryside chains include an optionally substituted alkyl, aryl, heterocyclyl,alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.Exemplary amino acids include alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline,ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine,taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groupsmay be optionally substituted with one, two, three, or, in the case ofamino acid groups of two carbons or more, four substituentsindependently selected from the group consisting of: (1) C₁₋₆ alkoxy;(2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy;(9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(1-1′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aminoalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aminoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, indenyl, and the like, and may be optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from the groupconsisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy;(14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy);(17) —(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, andR^(A′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b)C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integer fromzero to four and where R^(D′) is selected from the group consisting of(a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁₋₆alkaryl or a C₁-alkheterocyclyl can be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The term “arylalkoxy,” as used herein, represents an alkaryl group, asdefined herein, attached to the parent molecular group through an oxygenatom. Exemplary unsubstituted alkoxyalkyl groups include from 7 to 30carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₆₋₁₀aryl-C₁₋₆ alkoxy, C₆₋₁₀ aryl-C₁₋₁₀ alkoxy, or C₆₋₁₀ aryl-C₁₋₂₀ alkoxy).In some embodiments, the arylalkoxy group can be substituted with 1, 2,3, or 4 substituents as defined herein

The term “aryloxy” represents a chemical substituent of formula —OR′,where R′ is an aryl group of 6 to 18 carbons, unless otherwisespecified. In some embodiments, the aryl group can be substituted with1, 2, 3, or 4 substituents as defined herein.

The term “aryloyl,” as used herein, represents an aryl group, as definedherein, that is attached to the parent molecular group through acarbonyl group. Exemplary unsubstituted aryloyl groups are of 7 to 11carbons. In some embodiments, the aryl group can be substituted with 1,2, 3, or 4 substituents as defined herein.

The term “azido” represents an —N₃ group, which can also be representedas —N═N═N.

The term “bicyclic,” as used herein, refer to a structure having tworings, which may be aromatic or non-aromatic. Bicyclic structuresinclude spirocyclyl groups, as defined herein, and two rings that shareone or more bridges, where such bridges can include one atom or a chainincluding two, three, or more atoms. Exemplary bicyclic groups include abicyclic carbocyclyl group, where the first and second rings arecarbocyclyl groups, as defined herein; a bicyclic aryl groups, where thefirst and second rings are aryl groups, as defined herein; bicyclicheterocyclyl groups, where the first ring is a heterocyclyl group andthe second ring is a carbocyclyl (e.g., aryl) or heterocyclyl (e.g.,heteroaryl) group; and bicyclic heteroaryl groups, where the first ringis a heteroaryl group and the second ring is a carbocyclyl (e.g., aryl)or heterocyclyl (e.g., heteroaryl) group. In some embodiments, thebicyclic group can be substituted with 1, 2, 3, or 4 substituents asdefined herein for cycloalkyl, heterocyclyl, and aryl groups.

The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to anoptionally substituted C₃₋₁₂ monocyclic, bicyclic, or tricyclicstructure in which the rings, which may be aromatic or non-aromatic, areformed by carbon atoms. Carbocyclic structures include cycloalkyl,cycloalkenyl, and aryl groups.

The term “carbamoyl,” as used herein, represents —C(O)—N(R^(N1))₂, wherethe meaning of each R^(N1) is found in the definition of “amino”provided herein.

The term “carbamoylalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carbamoyl group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “carbamyl,” as used herein, refers to a carbamate group havingthe structure

—NR^(N1)C(═O)OR or —OC(═O)N(R^(N1))₂, where the meaning of each R^(N1)is found in the definition of “amino” provided herein, and R is alkyl,cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl (e.g.,heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as definedherein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxyaldehyde” represents an acyl group having the structure—CHO.

The term “carboxy,” as used herein, means —CO₂H.

The term “carboxyalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkoxy group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein for the alkyl group.

The term “carboxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkoxy” represents a chemical substituent of formula —OR,where R is a C₃₋₈ cycloalkyl group, as defined herein, unless otherwisespecified. The cycloalkyl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein. Exemplary unsubstitutedcycloalkoxy groups are from 3 to 8 carbons. In some embodiment, thecycloalkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl,and the like. When the cycloalkyl group includes one carbon-carbondouble bond, the cycloalkyl group can be referred to as a “cycloalkenyl”group. Exemplary cycloalkenyl groups include cyclopentenyl,cyclohexenyl, and the like. The cycloalkyl groups of this invention canbe optionally substituted with: (1) C₁₋₇ acyl (e.g., carboxyaldehyde);(2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl,(carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g., perfluoroalkyl),hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3)C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such as perfluoroalkoxy); (4) C₁₋₆alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8)azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo;(12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g.,C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q is an integer fromzero to four, and R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl;(18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to fourand where R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d)C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integerfrom zero to four and where R^(D′) is selected from the group consistingof (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20)(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “effective amount” of an agent, as used herein, is that amountsufficient to effect beneficial or desired results, for example,clinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that treats cancer, an effective amount of anagent is, for example, an amount sufficient to achieve treatment, asdefined herein, of cancer, as compared to the response obtained withoutadministration of the agent.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “haloalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkoxy may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkoxy groupsinclude perfluoroalkoxys (e.g., —OCF₃), —OCHF₂, —OCH₂F, —OCC₁₃,—OCH₂CH₂Br, —OCH₂CH(CH₂CH₂Br)CH₃, and —OCHICH₃. In some embodiments, thehaloalkoxy group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “haloalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkyl may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkyl groupsinclude perfluoroalkyls (e.g., —CF₃), —CHF₂, —CH₂F, —CCl₃, —CH₂CH₂Br,—CH₂CH(CH₂CH₂Br)CH₃, and —CHICH₃. In some embodiments, the haloalkylgroup can be further substituted with 1, 2, 3, or 4 substituent groupsas described herein for alkyl groups.

The term “heteroalkylene,” as used herein, refers to an alkylene group,as defined herein, in which one or two of the constituent carbon atomshave each been replaced by nitrogen, oxygen, or sulfur. In someembodiments, the heteroalkylene group can be further substituted with 1,2, 3, or 4 substituent groups as described herein for alkylene groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like. Examples of fused heterocyclyls includetropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics includepyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl,morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl;2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl;3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where

E′ is selected from the group consisting of —N— and —CH—; F′ is selectedfrom the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—, —CH═N—,—CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—, —O—, and—S—; and G′ is selected from the group consisting of —CH— and —N—. Anyof the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇ acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and RF, is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “(heterocyclyl)imino,” as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an imino group. In some embodiments, the heterocyclylgroup can be substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “(heterocyclyl)oxy,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group throughan oxygen atom. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “(heterocyclyl)oyl,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group througha carbonyl group. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxy,” as used herein, represents an —OH group.

The term “hydroxyalkenyl,” as used herein, represents an alkenyl group,as defined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by dihydroxypropenyl,hydroxyisopentenyl, and the like.

The term “hydroxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by hydroxymethyl,dihydroxypropyl, and the like.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3rd Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups, such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “nitro,” as used herein, represents an —NO₂ group.

The term “oxo” as used herein, represents ═O.

The term “perfluoroalkyl,” as used herein, represents an alkyl group, asdefined herein, where each hydrogen radical bound to the alkyl group hasbeen replaced by a fluoride radical. Perfluoroalkyl groups areexemplified by trifluoromethyl, pentafluoroethyl, and the like.

The term “perfluoroalkoxy,” as used herein, represents an alkoxy group,as defined herein, where each hydrogen radical bound to the alkoxy grouphas been replaced by a fluoride radical. Perfluoroalkoxy groups areexemplified by trifluoromethoxy, pentafluoroethoxy, and the like.

The term “spirocyclyl,” as used herein, represents a C₂₋₇ alkylenediradical, both ends of which are bonded to the same carbon atom of theparent group to form a spirocyclic group, and also a C₁₋₆ heteroalkylenediradical, both ends of which are bonded to the same atom. Theheteroalkylene radical forming the spirocyclyl group can containing one,two, three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. In some embodiments, thespirocyclyl group includes one to seven carbons, excluding the carbonatom to which the diradical is attached. The spirocyclyl groups of theinvention may be optionally substituted with 1, 2, 3, or 4 substituentsprovided herein as optional substituents for cycloalkyl and/orheterocyclyl groups.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

The term “sulfoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a sulfo group of —SO₃H. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thioalkaryl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkaryl group. In someembodiments, the alkaryl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein.

The term “thioalkheterocyclyl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkheterocyclyl group. In someembodiments, the alkheterocyclyl group can be further substituted with1, 2, 3, or 4 substituent groups as described herein.

The term “thioalkoxy,” as used herein, represents a chemical substituentof formula —SR, where R is an alkyl group, as defined herein. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “thiol” represents an —SH group.

Compound: As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone-enol pairs, amide-imidicacid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Condition: As used herein, the term “condition” refers to a disorderthat presents with observable symptoms.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an oligonucleotide orpolypeptide or may apply to a portion, region or feature thereof.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the engineered RNA or mRNA of the present invention may be singleunits or multimers or comprise one or more components of a complex orhigher order structure.

Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing,suppressing the growth, division, or multiplication of a cell (e.g., amammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of anoncology-related polynucleotide, oncology-related primary construct oroncology-related mmRNA to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Disease: As used herein, the term “disease” refers to an abnormalcondition affecting the body of an organism often showing specificbodily symptoms.

Disorder: As used herein, the term “disorder, “refers to a disruption ofor an interference with normal functions or established systems of thebody.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Dosing regimen: As used herein, a “dosing regimen” is a schedule ofadministration or physician determined regimen of treatment,prophylaxis, or palliative care.

Dose splitting factor (DSF)-ratio of PUD of dose split treatment dividedby PUD of total daily dose or single unit dose. The value is derivedfrom comparison of dosing regimens groups.

Encoded protein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence which encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Exosome: As used herein, “exosome” is a vesicle secreted by mammaliancells or a complex involved in RNA degradation.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least anoncology-related polynucleotide, oncology-related primary construct oroncology-related mmRNA and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Genotype: As used herein, “genotype” refers to the change in thegenotype, or genetic makeup, of a subject, cell, tissue, organ and/ororganism.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the invention, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the invention, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between oligonucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof. Methods forisolating compounds and their salts are routine in the art.

Linker: As used herein, a linker refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker may be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form mmRNA multimers (e.g., throughlinkage of two or more oncology-related polynucleotides,oncology-related primary constructs, or oncology-related mmRNAmolecules) or mmRNA conjugates, as well as to administer a payload, asdescribed herein. Examples of chemical groups that can be incorporatedinto the linker include, but are not limited to, alkyl, alkenyl,alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers,Other examples include, but are not limited to, cleavable moietieswithin the linker, such as, for example, a disulfide bond (—S—S—) or anazo bond (—N═N—), which can be cleaved using a reducing agent orphotolysis. Non-limiting examples of a selectively cleavable bondinclude an amido bond can be cleaved for example by the use oftris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/orphotolysis, as well as an ester bond can be cleaved for example byacidic or basic hydrolysis.

Metastasis: As used herein, the term “metastasis” means the process bywhich cancer spreads from the place at which it first arose as a primarytumor to distant locations in the body.

Method of Treating: The phrase “a method of treating” or its equivalent,when applied to, for example, cancer refers to a procedure or course ofaction that is designed to reduce or eliminate the number of cancercells, prevent the increase in the number of cancer cells, or toalleviate the symptoms of a cancer in a subject. A method of treatingcancer or another oncology-related disorder does not necessarily meanthat the cancer cells or other disorder will, in fact, be completelyeliminated, that the number of cells or disorder will, in fact, bereduced, or that the symptoms of a cancer or other disorder will, infact, be alleviated. Often, a method of treating cancer will beperformed even with a low likelihood of success, but which, given themedical history and estimated survival expectancy of a subject, isnevertheless deemed an overall beneficial course of action.

MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA)binding site represents a nucleotide location or region of a nucleicacid transcript to which at least the “seed” region of a miRNA binds.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the invention. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present invention are modified bythe introduction of non-natural nucleosides and/or nucleotides, e.g., asit relates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “modified”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Mucus: As used herein, “mucus” refers to the natural substance that isviscous and comprises mucin glycoproteins.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Non-human vertebrate: As used herein, a “non human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Oncology-related: As used herein, the term “oncology-related” refers toany disease, disorder, condition, treatment, process, substance orcompound related to any aspect of one or more hyperproliferativediseases, disorders and/or conditions including, but not limited to,cancer.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Paratope: As used herein, a “paratope” refers to the antigen-bindingsite of an antibody.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g. alkyl) per se is optional.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Pharmaceutical composition: The phrase “pharmaceutical composition”refers to a composition that alters the etiology of a disease, disorderand/or condition.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, andUse, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge etal., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of whichis incorporated herein by reference in its entirety.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Phenotype: As used herein, “phenotype” refers to the set of observablecharacteristics of a subject, cell, tissue, organ and/or organism.

Polypeptide per unit drug (PUD): As used herein, a PUD or product perunit drug, is defined as a subdivided portion of total daily dose,usually 1 mg, pg, kg, etc., of a product (such as a polypeptide) asmeasured in body fluid or tissue, usually defined in concentration suchas pmol/mL, mmol/mL, etc divided by the measure in the body fluid.

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestered in some wayand which release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Progression: As used herein, the term “progression” (e.g., cancerprogression) means the advancement or worsening of or toward a diseaseor condition.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Pseudouridine: As used herein, pseudouridine refers to the C-glycosideisomer of the nucleoside uridine. A “pseudouridine analog” is anymodification, variant, isoform or derivative of pseudouridine. Forexample, pseudouridine analogs include but are not limited to1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine,1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine,1-methylpseudouridine (m¹ψ), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine,1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ), and2′-O-methyl-pseudouridine (ψm).

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Regression: As used herein, the term “regression” or “degree ofregression” refers to the reversal, either phenotypically orgenotypically, of a cancer progression. Slowing or stopping cancerprogression may be considered regression.

Reducing the effect: As used herein, the phrase “reducing the effect”when referring to symptoms, means reducing, eliminating or alleviatingthe symptom in the subject. It does not necessarily mean that thesymptom will, in fact, be completely eliminated, reduced or alleviated.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Side effect: As used herein, the phrase “side effect” refers to asecondary effect of treatment.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence which can direct the transport or localization of aprotein.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Skin: The term “skin” is the thin layer of tissue forming the naturalouter covering of the body of a subject and includes the epidermis andthe dermis. The dermis is the thick layer of living tissue below theepidermis which is the surface epithelium of the skin.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Symptom: As used herein, the term “symptom” is a signal of a disease,disorder and/or condition. For example, symptoms may be felt or noticedby the subject who has them but may not be easily accessed by looking ata subject's outward appearance or behaviors. Examples of symptomsinclude, but are not limited to, weakness, aches and pains, fever,fatigue, weight loss, blood clots, increased blood calcium levels, lowwhite blood cell count, short of breath, dizziness, headaches,hyperpigmentation, jaundice, erthema, pruritis, excessive hair growth,change in bowel habits, change in bladder function, long-lasting sores,white patches inside the mouth, white spots on the tongue, unusualbleeding or discharge, thickening or lump on parts of the body,indigestion, trouble swallowing, changes in warts or moles, change innew skin and nagging cough or hoarseness.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Tumor: As used herein, a “tumor” is an abnormal growth of tissue,whether benign or malignant.

Tumor growth: As used herein, the term “tumor growth” or “tumormetastases” means an increased mass or volume of the tumor or expansionof the tumor distribution.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1 Modified mRNA Production

Modified mRNAs (mmRNA) according to the invention may be made usingstandard laboratory methods and materials. The open reading frame (ORF)of the gene of interest may be flanked by a 5′ untranslated region (UTR)which may contain a strong Kozak translational initiation signal and/oran alpha-globin 3′ UTR which may include an oligo(dT) sequence fortemplated addition of a poly-A tail. The modified mRNAs may be modifiedto reduce the cellular innate immune response. The modifications toreduce the cellular response may include pseudouridine (ψ) and5-methyl-cytidine (5meC, 5mc or m⁵C). (See, Kariko K et al. Immunity23:165-75 (2005), Kariko K et al. Mol Ther 16:1833-40 (2008), Anderson BR et al. NAR (2010); each of which are herein incorporated by referencein their entireties).

The ORF may also include various upstream or downstream additions (suchas, but not limited to, β-globin, tags, etc.) may be ordered from anoptimization service such as, but limited to, DNA2.0 (Menlo Park,Calif.) and may contain multiple cloning sites which may have XbaIrecognition. Upon receipt of the construct, it may be reconstituted andtransformed into chemically competent E. coli.

The methods described herein to make modified mRNA may be used toproduce molecules of all sizes including long molecules. Modified mRNAusing the described methods has been made for different sized moleculesincluding glucosidase, alpha; acid (GAA) (3.2 kb), cystic fibrosistransmembrane conductance regulator (CFTR) (4.7 kb), Factor VII (7.3kb), lysosomal acid lipase (45.4 kDa), glucocerebrosidase (59.7 kDa) andiduronate 2-sulfatase (76 kDa).

For the present invention, NEB DH5-alpha Competent E. coli are used.Transformations are performed according to NEB instructions using 100 ngof plasmid. The protocol is as follows:

-   -   1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for        10 minutes.    -   2. Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell        mixture. Carefully flick the tube 4-5 times to mix cells and        DNA. Do not vortex.    -   3. Place the mixture on ice for 30 minutes. Do not mix.    -   4. Heat shock at 42° C. for exactly 30 seconds. Do not mix.    -   5. Place on ice for 5 minutes. Do not mix.    -   6. Pipette 950 μl of room temperature SOC into the mixture.    -   7. Place at 37° C. for 60 minutes. Shake vigorously (250 rpm) or        rotate.    -   8. Warm selection plates to 37° C.    -   9. Mix the cells thoroughly by flicking the tube and inverting.

Spread 50-100 μl of each dilution onto a selection plate and incubateovernight at 37° C. Alternatively, incubate at 30° C. for 24-36 hours or25° C. for 48 hours.

A single colony is then used to inoculate 5 ml of LB growth media usingthe appropriate antibiotic and then allowed to grow (250 RPM, 37° C.)for 5 hours. This is then used to inoculate a 200 ml culture medium andallowed to grow overnight under the same conditions.

To isolate the plasmid (up to 850 μg), a maxi prep is performed usingthe Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.),following the manufacturer's instructions.

In order to generate cDNA for In Vitro Transcription (IVT), the plasmid(an Example of which is shown in FIG. 3) is first linearized using arestriction enzyme such as XbaI. A typical restriction digest with XbaIwill comprise the following: Plasmid 1.0 μg; 10× Buffer 1.0 μl; XbaI 1.5μl; dH₂O up to 10 μl; incubated at 37° C. for 1 hr. If performing at labscale (<5 μg), the reaction is cleaned up using Invitrogen's PURELINK™PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions. Largerscale purifications may need to be done with a product that has a largerload capacity such as Invitrogen's standard PURELINK™ PCR Kit (Carlsbad,Calif.). Following the cleanup, the linearized vector is quantifiedusing the NanoDrop and analyzed to confirm linearization using agarosegel electrophoresis.

As a non-limiting example, G-CSF may represent the polypeptide ofinterest. Sequences used in the steps outlined in Examples 1-5 are shownin Table 12. It should be noted that the start codon (ATG) has beenunderlined in each sequence of Table 12.

TABLE 12 G-CSF Sequences SEQ ID NO Description 33894 cDNAsequence:ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGGCCACCCCGAGGAGCTGGTGCTGCTCGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCT TGCCCAGCCCTGA 33895cDNA having T7 polymerase site, AfeI and Xba restriction site:TAATACGACTCACTATA GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCAT GCATCTAGA 33896Optimized sequence; containing T7 poly-merase site, AfeI and Xba restriction site TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCACACCTGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCAT GCATCTAGA 33897mRNA sequence (transcribed) GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCU UUGAAUAAAGCCUGAGUAGGAAG

Example 2 PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2× KAPA ReadyMix 12.5 μl; Forward Primer (10 uM) 0.75 μl;Reverse Primer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH₂O dilutedto 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45sec, then 72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention incorporates a poly-T₁₂₀ fora poly-A₁₂₀ in the mRNA. Other reverse primers with longer or shorterpoly(T) tracts can be used to adjust the length of the poly(A) tail inthe mRNA.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NANODROP™ andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 3 In Vitro Transcription (IVT)

The in vitro transcription reaction generates mRNA containing modifiednucleotides or modified RNA. The input nucleotide triphosphate (NTP) mixis made in-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

1. Template cDNA 1.0 μg 2. 10x transcription buffer (400 mM Tris-HCl pH8.0, 2.0 μl 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 3. Custom NTPs(25 mM each) 7.2 μl 4. RNase Inhibitor  20 U 5. T7 RNA polymerase 3000 U6. dH₂0 Up to 20.0 μl. and 7. Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 4 Enzymatic Capping of mRNA

Capping of the mRNA is performed as follows where the mixture includes:IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixture is incubated at65° C. for 5 minutes to denature RNA, and then is transferredimmediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂O (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The mRNA is then purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.)following the manufacturer's instructions. Following the cleanup, theRNA is quantified using the NANODROP™ (ThermoFisher, Waltham, Mass.) andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred. The RNA productmay also be sequenced by running a reverse-transcription-PCR to generatethe cDNA for sequencing.

Example 5 PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis preferably a recombinant enzyme expressed in yeast.

For studies performed and described herein, the poly-A tail is encodedin the IVT template to comprise 160 nucleotides in length. However, itshould be understood that the processivity or integrity of the polyAtailing reaction may not always result in exactly 160 nucleotides. HencepolyA tails of approximately 160 nucleotides, e.g, about 150-165, 155,156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scopeof the invention.

Example 6 Natural 5′ Caps and 5′ Cap Analogues

5′-capping of modified RNA may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5)ppp(5′) G [the ARCA cap];G(5)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 7 Capping

A. Protein Expression Assay

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 33894;mRNA sequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 33897with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA (3′ O-Me-m7G(5′)ppp(5′)G) cap analog orthe Cap1 structure can be transfected into human primary keratinocytesat equal concentrations. 6, 12, 24 and 36 hours post-transfection theamount of G-CSF secreted into the culture medium can be assayed byELISA. Synthetic mRNAs that secrete higher levels of G-CSF into themedium would correspond to a synthetic mRNA with a highertranslationally-competent Cap structure.

B. Purity Analysis Synthesis

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 33894;mRNA sequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 33897with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA cap analog or the Cap1 structure crudesynthesis products can be compared for purity using denaturingAgarose-Urea gel electrophoresis or HPLC analysis. Synthetic mRNAs witha single, consolidated band by electrophoresis correspond to the higherpurity product compared to a synthetic mRNA with multiple bands orstreaking bands. Synthetic mRNAs with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure mRNA population.

C. Cytokine Analysis

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 33894;mRNA sequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 33897with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can betransfected into human primary keratinocytes at multiple concentrations.6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatorycytokines such as TNF-alpha and IFN-beta secreted into the culturemedium can be assayed by ELISA. Synthetic mRNAs that secrete higherlevels of pro-inflammatory cytokines into the medium would correspond toa synthetic mRNA containing an immune-activating cap structure.

D. Capping Reaction Efficiency

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 33894;mRNA sequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 33897with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can beanalyzed for capping reaction efficiency by LC-MS after capped mRNAnuclease treatment. Nuclease treatment of capped mRNAs would yield amixture of free nucleotides and the capped 5′-5-triphosphate capstructure detectable by LC-MS. The amount of capped product on the LC-MSspectra can be expressed as a percent of total mRNA from the reactionand would correspond to capping reaction efficiency. The cap structurewith higher capping reaction efficiency would have a higher amount ofcapped product by LC-MS.

Example 8 Agarose Gel Electrophoresis of Modified RNA or RT PCR Products

Individual modified RNAs (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) are loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes according to the manufacturer protocol.

Example 9 Nanodrop Modified RNA Quantification and UV Spectral Data

Modified RNAs in TE buffer (1 μl) are used for Nanodrop UV absorbancereadings to quantitate the yield of each modified RNA from an in vitrotranscription reaction.

Example 10 Formulation of Modified mRNA Using Lipidoids

Modified mRNAs (mmRNA) are formulated for in vitro experiments by mixingthe mmRNA with the lipidoid at a set ratio prior to addition to cells.In vivo formulation may require the addition of extra ingredients tofacilitate circulation throughout the body. To test the ability of theselipidoids to form particles suitable for in vivo work, a standardformulation process used for siRNA-lipidoid formulations was used as astarting point. Initial mmRNA-lipidoid formulations may consist ofparticles composed of 42% lipidoid, 48% cholesterol and 10% PEG, withfurther optimization of ratios possible. After formation of theparticle, mmRNA is added and allowed to integrate with the complex. Theencapsulation efficiency is determined using a standard dye exclusionassays.

Materials and Methods for Examples 11-15

A. Lipid Synthesis

Six lipids, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 andDLin-MC3-DMA, were synthesized by methods outlined in the art in orderto be formulated with modified RNA. DLin-DMA and precursors weresynthesized as described in Heyes et. al, J. Control Release, 2005, 107,276-287. DLin-K-DMA and DLin-KC2-DMA and precursors were synthesized asdescribed in Semple et. al, Nature Biotechnology, 2010, 28, 172-176.98N12-5 and precursor were synthesized as described in Akinc et. al,Nature Biotechnology, 2008, 26, 561-569.

C12-200 and precursors were synthesized according to the method outlinedin Love et. al, PNAS, 2010, 107, 1864-1869. 2-epoxydodecane (5.10 g,27.7 mmol, 8.2 eq) was added to a vial containing Amine 200 (0.723 g,3.36 mmol, 1 eq) and a stirring bar. The vial was sealed and warmed to80° C. The reaction was stirred for 4 days at 80° C. Then the mixturewas purified by silica gel chromatography using a gradient from puredichloromethane (DCM) to DCM:MeOH 98:2. The target compound was furtherpurified by RP-HPLC to afford the desired compound.

DLin-MC3-DMA and precursors were synthesized according to proceduresdescribed in WO 2010054401 herein incorporated by reference in itsentirety. A mixture of dilinoleyl methanol (1.5 g, 2.8 mmol, 1 eq),N,N-dimethylaminobutyric acid (1.5 g, 2.8 mmol, 1 eq), DIPEA (0.73 mL,4.2 mmol, 1.5 eq) and TBTU (1.35 g, 4.2 mmol, 1.5 eq) in 10 mL of DMFwas stirred for 10 h at room temperature. Then the reaction mixture wasdiluted in ether and washed with water. The organic layer was dried overanhydrous sodium sulfate, filtrated and concentrated under reducedpressure. The crude product was purified by silica gel chromatographyusing a gradient DCM to DCM:MeOH 98:2. Subsequently the target compoundwas subjected to an additional RP-HPLC purification which was done usinga YMC-Pack C4 column to afford the target compound.

B. Formulation of Modified RNA Nanoparticles

Solutions of synthesized lipid, 1,2-distearoyl-3-phosphatidylcholine(DSPC) (Avanti Polar Lipids, Alabaster, Ala.), cholesterol(Sigma-Aldrich, Taufkirchen, Germany), andα-[3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-ω-methoxy-polyoxyethylene(PEG-c-DOMG) (NOF, Bouwelven, Belgium) were prepared at concentrationsof 50 mM in ethanol and stored at −20° C. The lipids were combined toyield molar ratio of 50:10:38.5:1.5 (Lipid:DSPC:Cholesterol:PEG-c-DOMG)and diluted with ethanol to a final lipid concentration of 25 mM.Solutions of modified mRNA at a concentration of 1-2 mg/mL in water werediluted in 50 mM sodium citrate buffer at a pH of 3 to form a stockmodified mRNA solution. Formulations of the lipid and modified mRNA wereprepared by combining the synthesized lipid solution with the modifiedmRNA solution at total lipid to modified mRNA weight ratio of 10:1,15:1, 20:1 and 30:1. The lipid ethanolic solution was rapidly injectedinto aqueous modified mRNA solution to afford a suspension containing33% ethanol. The solutions were injected either manually (MI) or by theaid of a syringe pump (SP) (Harvard Pump 33 Dual Syringe Pump HarvardApparatus Holliston, Mass.).

To remove the ethanol and to achieve the buffer exchange, theformulations were dialyzed twice against phosphate buffered saline(PBS), pH 7.4 at volumes 200-times of the primary product using aSlide-A-Lyzer cassettes (Thermo Fisher Scientific Inc. Rockford, Ill.)with a molecular weight cutoff (MWCO) of 10 kD. The first dialysis wascarried at room temperature for 3 hours and then the formulations weredialyzed overnight at 4° C. The resulting nanoparticle suspension wasfiltered through 0.2 μm sterile filter (Sarstedt, Nümbrecht, Germany)into glass vials and sealed with a crimp closure.

C. Characterization of Formulations

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) was used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the modified mRNA nanoparticles in 1×PBSin determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy was used to determine the concentrationof modified mRNA nanoparticle formulation. 100 μL of the dilutedformulation in 1×PBS was added to 900 μL of a 4:1 (v/v) mixture ofmethanol and chloroform. After mixing, the absorbance spectrum of thesolution was recorded between 230 nm and 330 nm on a DU 800spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea,Calif.). The modified RNA concentration in the nanoparticle formulationwas calculated based on the extinction coefficient of the modified RNAused in the formulation and on the difference between the absorbance ata wavelength of 260 nm and the baseline value at a wavelength of 330 nm.

QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.)was used to evaluate the encapsulation of modified RNA by thenanoparticle. The samples were diluted to a concentration ofapproximately 5 μg/mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).50 μL of the diluted samples were transferred to a polystyrene 96 wellplate, then either 50 μL of TE buffer or 50 μL of a 2% Triton X-100solution was added. The plate was incubated at a temperature of 37° C.for 15 minutes. The RIBOGREEN® reagent was diluted 1:100 in TE buffer,100 μL of this solution was added to each well. The fluorescenceintensity was measured using a fluorescence plate reader (Wallac Victor1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) at an excitationwavelength of ˜480 nm and an emission wavelength of ˜520 nm. Thefluorescence values of the reagent blank were subtracted from that ofeach of the samples and the percentage of free modified RNA wasdetermined by dividing the fluorescence intensity of the intact sample(without addition of Triton X-100) by the fluorescence value of thedisrupted sample (caused by the addition of Triton X-100).

D. In Vitro Incubation

Human embryonic kidney epithelial (HEK293) and hepatocellular carcinomaepithelial (HepG2) cells (LGC standards GmbH, Wesel, Germany) wereseeded on 96-well plates (Greiner Bio-one GmbH, Frickenhausen, Germany)and plates for HEK293 cells were precoated with collagen type1. HEK293were seeded at a density of 30,000 and HepG2 were seeded at a density of35,000 cells per well in 100 μl cell culture medium. For HEK293 the cellculture medium was DMEM, 10% FCS, adding 2 mM L-Glutamine, 1 mMSodiumpyruvate and 1× non-essential amino acids (Biochrom AG, Berlin,Germany) and 1.2 mg/ml Sodiumbicarbonate (Sigma-Aldrich, Munich,Germany) and for HepG2 the culture medium was MEM (Gibco LifeTechnologies, Darmstadt, Germany), 10% FCS adding 2 mM L-Glutamine, 1 mMSodiumpyruvate and 1× non-essential amino acids (Biochrom AG, Berlin,Germany. Formulations containing mCherry mRNA (mRNA sequence shown inSEQ ID NO: 33898; polyA tail of approximately 160 nucleotides not shownin sequence; 5′cap, Cap1) were added in quadruplicates directly afterseeding the cells and incubated. The mCherry cDNA with the T7 promoter,5′untranslated region (UTR) and 3′ UTR used in in vitro transcription(IVT) is given in SEQ ID NO: 33899. The mCherry mRNA was modified with a5meC at each cytosine and pseudouridine replacement at each uridinesite.

Cells were harvested by transferring the culture media supernatants to a96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg,Germany). Cells were trypsinized with 1/2 volume Trypsin/EDTA (BiochromAG, Berlin, Germany), pooled with respective supernatants and fixed byadding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5%formaldehyde (Merck, Darmstadt, Germany). Samples then were submitted toa flow cytometer measurement with a 532 nm excitation laser and the610/20 filter for PE-Texas Red in a LSRII cytometer (Beckton DickinsonGmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI) of allevents and the standard deviation of four independent wells arepresented in for samples analyzed.

Example 11 Purification of Nanoparticle Formulations

Nanoparticle formulations of DLin-KC2-DMA and 98N12-5 in HEK293 andHepG2 were tested to determine if the mean fluorescent intensity (MFI)was dependent on the lipid to modified RNA ratio and/or purification.Three formulations of DLin-KC2-DMA and two formulations of 98N12-5 wereproduced using a syringe pump to the specifications described in Table13. Purified samples were purified by SEPHADEX™ G-25 DNA grade (GEHealthcare, Sweden). Each formulation before and after purification (aP)was tested at concentration of 250 ng modified RNA per well in a 24 wellplate. The percentage of cells that are positive for the marker for FL4channel (% FL4-positive) when analyzed by the flow cytometer for eachformulation and the background sample, and the MFI of the marker for theFL4 channel for each formulation and the background sample are shown inTable 14. The formulations which had been purified had a slightly higherMFI than those formulations tested before purification.

TABLE 13 Formulations Formulation # Lipid Lipid/RNA wt/wt Mean size (nm)NPA-001-1 DLin-KC2-DMA 10 155 nm PDI: 0.08 NPA-001-1 aP DLin-KC2-DMA 10141 nm PDI: 0.14 NPA-002-1 DLin-KC2-DMA 15 140 nm PDI: 0.11 NPA-002-1 aPDLin-KC2-DMA 15 125 nm PDI: 0.12 NPA-003-1 DLin-KC2-DMA 20 114 nm PDI:0.08 NPA-003-1 aP DLin-KC2-DMA 20 104 nm PDI: 0.06 NPA-005-1 98N12-5 15127 nm PDI: 0.12 NPA-005-1 aP 98N12-5 15 134 nm PDI: 0.17 NPA-006-198N12 20 126 nm PDI: 0.08 NPA-006-1 aP 98N12 20 118 nm PDI: 0.13

TABLE 14 HEK293 and HepG2, 24-well, 250 ng Modified RNA/well %FL4-positive FL4 MFI Formulation HEK293 HepG2 HEK293 HepG2 Untreated0.33 0.40 0.25 0.30 NPA-001-1 62.42 5.68 1.49 0.41 NPA-001-ap 87.32 9.023.23 0.53 NPA-002-1 91.28 9.90 4.43 0.59 NPA-002-ap 92.68 14.02 5.070.90 NPA-003-1 87.70 11.76 6.83 0.88 NPA-003-ap 88.88 15.46 8.73 1.06NPA-005-1 50.60 4.75 1.83 0.46 NPA-005-ap 38.64 5.16 1.32 0.46 NPA-006-154.19 13.16 1.30 0.60 NPA-006-ap 49.97 13.74 1.27 0.61

Example 12 Concentration Response Curve

Nanoparticle formulations of 98N12-5 (NPA-005) and DLin-KC2-DMA(NPA-003) were tested at varying concentrations to determine the MFI ofFL4 or mCherry (mRNA sequence shown in SEQ ID NO: 33898; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) over a range of doses.The formulations tested are outlined in Table 15. To determine theoptimal concentration of nanoparticle formulations of 98N12-5, varyingconcentrations of formulated modified RNA (100 ng, 10 ng, 1.0 ng, 0.1 ngand 0.01 ng per well) were tested in a 24-well plate of HEK293, and theresults of the FL4 MFI of each dose are shown in Table 16. Likewise, todetermine the optimal concentration of nanoparticle formulations ofDLin-KC2-DMA, varying concentrations of formulated modified RNA (250 ng100 ng, 10 ng, 1.0 ng, 0.1 ng and 0.01 ng per well) were tested in a24-well plate of HEK293, and the results of the FL4 MFI of each dose areshown in Table 17. Nanoparticle formulations of DLin-KC2-DMA were alsotested at varying concentrations of formulated modified RNA (250 ng, 100ng and 30 ng per well) in a 24 well plate of HEK293, and the results ofthe FL4 MFI of each dose are shown in Table 18. A dose of 1 ng/well for98N12-5 and a dose of 10 ng/well for DLin-KC2-DMA were found to resemblethe FL4 MFI of the background.

To determine how close the concentrations resembled the background, weutilized a flow cytometer with optimized filter sets for detection ofmCherry expression, and were able to obtain results with increasedsensitivity relative to background levels. Doses of 25 ng/well, 0.25ng/well, 0.025 ng/well and 0.0025 ng/well were analyzed for 98N12-5(NPA-005) and DLin-KC2-DMA (NPA-003) to determine the MFI of mCherry. Asshown in Table 19, the concentration of 0.025 ng/well and lesserconcentrations are similar to the background MFI level of mCherry whichis about 386.125.

TABLE 15 Formulations Formulation # NPA-003 NPA-005 Lipid DLin-KC2-98N12-5 DMA Lipid/RNA 20 15 wt/wt Mean size 114 nm 106 nm PDI: 0.08 PDI:0.12

TABLE 16 HEK293, NPA-005, 24-well, n = 4 Formulation FL4 MFI Untreatedcontrol 0.246 NPA-005 100 ng 2.2175 NPA-005 10 ng 0.651 NPA-005 1.0 ng0.28425 NPA-005 0.1 ng 0.27675 NPA-005 0.01 ng 0.2865

TABLE 17 HEK293, NPA-003, 24-well, n = 4 Formulation FL4 MFI Untreatedcontrol 0.3225 NPA-003 250 ng 2.9575 NPA-003 100 ng 1.255 NPA-003 10 ng0.40025 NPA-003 1 ng 0.33025 NPA-003 0.1 ng 0.34625 NPA-003 0.01 ng0.3475

TABLE 18 HEK293, NPA-003, 24-well, n = 4 Formulation FL4 MFI Untreatedcontrol 0.27425 NPA-003 250 ng 5.6075 NPA-003 100 ng 3.7825 NPA-003 30ng 1.5525

TABLE 19 Concentration and MFI MFI mCheny Formulation NPA-003 NPA-005   25 ng/well 11963.25 12256.75  0.25 ng/well 1349.75 2572.75  0.025ng/well 459.50 534.75 0.0025 ng/well 310.75 471.75

Example 13 Manual Injection and Syringe Pump Formulations

Two formulations of DLin-KC2-DMA and 98N12-5 were prepared by manualinjection (MI) and syringe pump injection (SP) and analyzed along with abackground sample to compare the MFI of mCherry (mRNA sequence shown inSEQ ID NO: 33898; polyA tail of approximately 160 nucleotides not shownin sequence; 5′cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) of the different formulations. Table 20 shows that thesyringe pump formulations had a higher MFI as compared to the manualinjection formulations of the same lipid and lipid/RNA ratio.

TABLE 20 Formulations and MFI Lipid/ Mean Method Formulation RNA size ofform- # Lipid wt/wt (nm) ulation MFI Untreated N/A N/A N/A N/A 674.67Control NPA-002 DLin-KC2- 15 140 nm MI 10318.25 DMA PDI: 0.11 NPA-002-2DLin-KC2- 15 105 nm SP 37054.75 DMA PDI: 0.04 NPA-003 DLin-KC2- 20 114nm MI 22037.5 DMA PDI: 0.08 NPA-003-2 DLin-KC2- 20  95 nm SP 37868.75DMA PDI: 0.02 NPA-005 98N12-5 15 127 nm MI 11504.75 PDI: 0.12 NPA-005-298N12-5 15 106 nm SP 9343.75 PDI: 0.07 NPA-006 98N12-5 20 126 nm MI11182.25 PDI: 0.08 NPA-006-2 98N12-5 20  93 nm SP 5167 PDI: 0.08

Example 14 Lipid Nanoparticle Formulations

Formulations of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 andDLin-MC3-DMA were incubated at a concentration of 60 ng/well or 62.5ng/well in a plate of HEK293 and 62.5 ng/well in a plate of HepG2 cellsfor 24 hours to determine the MFI of mCherry (SEQ ID NO: 33898; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) for eachformulation. The formulations tested are outlined in Table 21 below. Asshown in Table 22 for the 60 ng/well and Tables 23, 24 and 25 for the62.5 ng/well, the formulation of NPA-003 and NPA-018 have the highestmCherry MFI and the formulations of NPA-008, NPA-010 and NPA-013 aremost the similar to the background sample mCherry MFI value.

TABLE 21 Formulations Formulation Lipid/RNA Mean size # Lipid wt/wt (nm)NPA-001 DLin-KC2-DMA 10 155 nm PDI: 0.08 NPA-002 DLin-KC2-DMA 15 140 nmPDI: 0.11 NPA-002-2 DLin-KC2-DMA 15 105 nm PDI: 0.04 NPA-003DLin-KC2-DMA 20 114 nm PDI: 0.08 NPA-003-2 DLin-KC2-DMA 20 95 nm PDI:0.02 NPA-005 98N12-5 15 127 nm PDI: 0.12 NPA-006 98N12-5 20 126 nm PDI:0.08 NPA-007 DLin-DMA 15 148 nm PDI: 0.09 NPA-008 DLin-K-DMA 15 121 nmPDI: 0.08 NPA-009 C12-200 15 138 nm PDI: 0.15 NPA-010 DLin-MC3-DMA 15126 nm PDI: 0.09 NPA-012 DLin-DMA 20 86 nm PDI: 0.08 NPA-013 DLin-K-DMA20 104 nm PDI: 0.03 NPA-014 C12-200 20 101 nm PDI: 0.06 NPA-015DLin-MC3-DMA 20 109 nm PDI: 0.07

TABLE 22 HEK293, 96-well, 60 ng Modified RNA/well Formulation MFImCherry Untreated 871.81 NPA-001 6407.25 NPA-002 14995 NPA-003 29499.5NPA-005 3762 NPA-006 2676 NPA-007 9905.5 NPA-008 1648.75 NPA-009 2348.25NPA-010 4426.75 NPA-012 11466 NPA-013 2098.25 NPA-014 3194.25 NPA-01514524

TABLE 23 HEK293, 62.5 ng/well Formulation MFI mCherry Untreated 871.81NPA-001 6407.25 NPA-002 14995 NPA-003 29499.5 NPA-005 3762 NPA-006 2676NPA-007 9905.5 NPA-008 1648.75 NPA-009 2348.25 NPA-010 4426.75 NPA-01211466 NPA-013 2098.25 NPA-014 3194.25 NPA-015 14524

TABLE 24 HEK293, 62.5 ng/well Formulation MFI mCherry Untreated 295NPA-007 3504 NPA-012 8286 NPA-017 6128 NPA-003-2 17528 NPA-018 34142NPA-010 1095 NPA-015 5859 NPA-019 3229

TABLE 25 HepG2, 62.5 ng/well Formulation MFI mCherry Study 1 Untreated649.94 NPA-001 6006.25 NPA-002 8705 NPA-002-2 15860.25 NPA-003 15059.25NPA-003-2 28881 NPA-005 1676 NPA-006 1473 NPA-007 15678 NPA-008 2976.25NPA-009 961.75 NPA-010 3301.75 NPA-012 18333.25 NPA-013 5853 NPA-0142257 NPA-015 16225.75 Study 2 Untreated control 656 NPA-007 16798NPA-012 21993 NPA-017 20377 NPA-003-2 35651 NPA-018 40154 NPA-010 2496NPA-015 19741 NPA-019 16373

Example 15 In Vivo Formulation Studies

Rodents (n=5) are administered intravenously, subcutaneously orintramuscularly a single dose of a formulation containing a modifiedmRNA and a lipid. The modified mRNA administered to the rodents isselected from G-CSF (mRNA sequence shown in SEQ ID NO: 33897; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),erythropoietin (EPO) (mRNA sequence shown in SEQ ID NO: 33900; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1), Factor IX (mRNA sequence shown in SEQ ID NO: 33901; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) ormCherry (mRNA sequence shown in SEQ ID NO: 33898; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1). Theerythropoietin cDNA with the T7 promoter, 5′untranslated region (UTR)and 3′ UTR used in in vitro transcription (IVT) is given in SEQ ID NO:33902 and SEQ ID NO: 33903.

Each formulation also contains a lipid which is selected from one ofDLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA,reLNP, ATUPLEX®, DACC and DBTC. The rodents are injected with 100 ug, 10ug or 1 ug of the formulated modified mRNA and samples are collected atspecified time intervals.

Serum from the rodents administered formulations containing human G-CSFmodified mRNA are measured by specific G-CSF ELISA and serum from miceadministered human factor IX modified RNA is analyzed by specific factorIX ELISA or chromogenic assay. The liver and spleen from the miceadministered with mCherry modified mRNA are analyzed byimmunohistochemistry (IHC) or fluorescence-activated cell sorting(FACS). As a control, a group of mice are not injected with anyformulation and their serum and tissue are collected analyzed by ELISA,FACS and/or IHC.

A. Time Course

The rodents are administered formulations containing at least onemodified mRNA to study the time course of protein expression for theadministered formulation. The rodents are bled at specified timeintervals prior to and after administration of the modified mRNAformulations to determine protein expression and complete blood count.Samples are also collected from the site of administration of rodentsadministered modified mRNA formulations subcutaneously andintramuscularly to determine the protein expression in the tissue.

B. Dose Response

The rodents are administered formulations containing at least onemodified mRNA to determine dose response of each formulation. Therodents are bled at specified time intervals prior to and afteradministration of the modified mRNA formulations to determine proteinexpression and complete blood count. The rodents are also sacrificed toanalyze the effect of the modified mRNA formulation on the internaltissue. Samples are also collected from the site of administration ofrodents administered modified mRNA formulations subcutaneously andintramuscularly to determine the protein expression in the tissue.

C. Toxicity

The rodents are administered formulations containing at least onemodified mRNA to study toxicity of each formulation. The rodents arebled at specified time intervals prior to and after administration ofthe modified mRNA formulations to determine protein expression andcomplete blood count. The rodents are also sacrificed to analyze theeffect of the modified mRNA formulation on the internal tissue. Samplesare also collected from the site of administration of rodentsadministered modified mRNA formulations subcutaneously andintramuscularly to determine the protein expression in the tissue.

Example 16 PLGA Microsphere Formulations

Optimization of parameters used in the formulation of PLGA microspheresmay allow for tunable release rates and high encapsulation efficiencieswhile maintaining the integrity of the modified RNA encapsulated in themicrospheres. Parameters such as, but not limited to, particle size,recovery rates and encapsulation efficiency may be optimized to achievethe optimal formulation.

A. Synthesis of PLGA Microspheres

Polylacticglycolic acid (PLGA) microspheres were synthesized using thewater/oil/water double emulsification methods known in the art usingPLGA (Lactel, Cat #B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA),polyvinylalcohol (PVA) (Sigma, Cat #348406-25G, MW 13-23 k)dichloromethane and water. Briefly, 0.1 ml of water (W1) was added to 2ml of PLGA dissolved in dichloromethane (DCM) (O1) at concentrationsranging from 50-200 mg/ml of PLGA. The W1/O1 emulsion was homogenized(IKA Ultra-Turrax Homogenizer, T18) for 30 seconds at speed 4 (15,000rpm). The W1/O1 emulsion was then added to 100 to 200 ml of 0.3 to 1%PVA (W2) and homogenized for 1 minute at varied speeds. Formulationswere left to stir for 3 hours and then washed by centrifugation (20-25min, 4,000 rpm, 4° C.). The supernatant was discarded and the PLGApellets were resuspended in 5-10 ml of water, which was repeated 2×.Average particle size (represents 20-30 particles) for each formulationwas determined by microscopy after washing. Table 26 shows that anincrease in the PLGA concentration led to larger sized microspheres. APLGA concentration of 200 mg/mL gave an average particle size of 14.8μm, 100 mg/mL was 8.7 μm, and 50 mg/mL of PLGA gave an average particlesize of 4.0 μm.

TABLE 26 Varied PLGA Concentration PLGA PVA O1 Concen- W2 Concen-Average Sample Volume tration Volume tration Size ID (mL) (mg/mL) (mL)(%) Speed (μm) 1 2 200 100 0.3 5 14.8 2 2 100 100 0.3 5 8.7 3 2 50 1000.3 5 4.0

Table 27 shows that decreasing the homogenization speed from 5 (˜20,000rpm) to speed 4 (˜15,000 rpm) led to an increase in particle size from14.8 μm to 29.7 μm.

TABLE 27 Varied Homogenization Speed PLGA PVA O1 Concen- W2 Concen-Average Sample Volume tration Volume tration Size ID (mL) (mg/mL) (mL)(%) Speed (μm) 1 2 200 100 0.3 5 14.8 4 2 200 100 0.3 4 29.7

Table 28 shows that increasing the W2 volume (i.e. increasing the ratioof W2:O1 from 50:1 to 100:1), decreased average particle size slightly.Altering the PVA concentration from 0.3 to 1 wt % had little impact onPLGA microsphere size.

TABLE 28 Varied W2 Volume and Concentration PLGA PVA O1 Concen- W2Concen- Average Sample Volume tration Volume tration Size ID (mL)(mg/mL) (mL) (%) Speed (μm) 1 2 200 100 0.3 5 14.8 5 2 200 200 0.3 511.7 6 2 200 190 0.3 5 11.4 7 2 200 190 1.0 5 12.3

B. Encapsulation of Modified mRNA

Modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 33897; polyA tailof approximately 160 nucleotides not shown in sequence; 5′cap, Cap1;fully modified with 5-methylcytosine and pseudouridine) was dissolved inwater at a concentration of 2 mg/ml (W3). Three batches of PLGAmicrosphere formulations were made as described above with the followingparameters: 0.1 ml of W3 at 2 mg/ml, 1.6 ml of O1 at 200 mg/ml, 160 mlof W2 at 1%, and homogenized at a speed of 4 for the first emulsion(W3/O1) and homogenized at a speed of 5 for the second emulsion(W3/O1/W2). After washing by centrifugation, the formulations werefrozen in liquid nitrogen and then lyophilized for 3 days. To test theencapsulation efficiency of the formulations, the lyophilized materialwas deformulated in DCM for 6 hours followed by an overnight extractionin water. The modified RNA concentration in the samples was thendetermined by OD260. Encapsulation efficiency was calculated by takingthe actual amount of modified RNA and dividing by the starting amount ofmodified RNA. In the three batches tested, there was an encapsulationefficiency of 59.2, 49.8 and 61.3.

C. Integrity of Modified mRNA Encapsulated in PLGA Microspheres

Modified Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 33901; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) wasdissolved in water at varied concentrations (W4) to vary the weightpercent loading in the formulation (mg modified RNA/mg PLGA*100) and todetermine encapsulation efficiency. The parameters in Table 28 were usedto make four different batches of PLGA microsphere formulations with ahomogenization speed of 4 for the first emulsion (W4/O1) and ahomogenization speed of 5 for the second emulsion (W4/O1/W2).

TABLE 28 Factor IX PLGA Microsphere Formulation Parameters Factor WeightW4 Factor IX IX O1 PLGA W2 PVA % Volume Concentration Amount VolumeConcentration Volume Concentration (wt %) ID (uL) (mg/ml) (ug) (ml)(mg/ml) (ml) (%) Loading A 100 2.0 200.0 2.0 200 200 1.0 0.05 B 100 4.0400.0 2.0 200 200 1.0 0.10 C 400 2.0 800.0 2.0 200 200 1.0 0.20 D 4004.0 1600.0 2.0 200 200 1.0 0.40

After lyophilization, PLGA microspheres were weighed out in 2 mleppendorf tubes to correspond to ˜10 ug of modified RNA. Lyophilizationwas found to not destroy the overall structure of the PLGA microspheres.To increase weight percent loading (wt %) for the PLGA microspheres,increasing amounts of modified RNA were added to the samples. PLGAmicrospheres were deformulated by adding 1.0 ml of DCM to each tube andthen shaking the samples for 6 hours. For modified RNA extraction, 0.5ml of water was added to each sample and the samples were shakenovernight before the concentration of modified RNA in the samples wasdetermined by OD260. To determine the recovery of the extractionprocess, unformulated Factor IX modified mRNA (mRNA sequence shown inSEQ ID NO: 33901; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) (deformulation control) was spiked into DCM and wassubjected to the deformulation process. Table 29 shows the loading andencapsulation efficiency for the samples. All encapsulation efficiencysamples were normalized to the deformulation control.

TABLE 29 Weight Percent Loading and Encapsulation Efficiency TheoreticalActual modified Encapsulation modified RNA RNA Efficiency ID loading (wt%) loading (wt %) (%) A 0.05 0.06 97.1 B 0.10 0.10 85.7 C 0.20 0.18 77.6D 0.40 0.31 68.1 Control — — 100.0

D. Release Study of Modified mRNA Encapsulated in PLGA Microspheres

PLGA microspheres formulated with Factor IX modified mRNA (mRNA sequenceshown in SEQ ID NO: 33901; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) were deformulated as described aboveand the integrity of the extracted modified RNA was determined byautomated electrophoresis (Bio-Rad Experion). The extracted modifiedmRNA was compared against unformulated modified mRNA and thedeformulation control in order to test the integrity of the encapsulatedmodified mRNA. As shown in FIG. 4, the majority of modRNA was intact forbatch ID A, B, C and D, for the deformulated control (Deform control)and the unformulated control (Unform control).

E. Protein Expression of Modified mRNA Encapsulated in PLGA Microspheres

PLGA microspheres formulated with Factor IX modified mRNA (mRNA sequenceshown in SEQ ID NO: 33901; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) were deformulated as described aboveand the protein expression of the extracted modified RNA was determinedby an in vitro transfection assay. HEK293 cells were reverse transfectedwith 250 ng of Factor IX modified RNA complexed with RNAiMAX(Invitrogen) in triplicate.

Factor IX modified RNA was diluted in nuclease-free water to aconcentration of 25 ng/μl and RNAiMAX was diluted 13.3× in serum-freeEMEM. Equal volumes of diluted modified RNA and diluted RNAiMAX weremixed together and were allowed to stand for 20 to 30 minutes at roomtemperature. Subsequently, 20 μl of the transfection mix containing 250ng of Factor IX modified RNA was added to 80 μl of a cell suspensioncontaining 30,000 cells. Cells were then incubated for 16 h in ahumidified 37° C./5% CO2 cell culture incubator before harvesting thecell culture supernatant. Factor IX protein expression in the cellsupernatant was analyzed by an ELISA kit specific for Factor IX(Molecular Innovations, Cat #HFIXKT-TOT) and the protein expression isshown in Table 30. In all PLGA microsphere batches tested, Factor IXmodified RNA remained active and expressed Factor IX protein afterformulation in PLGA microspheres and subsequent deformulation.

TABLE 30 Protein Expression Factor IX Protein Sample Expression (ng/ml)Batch A 0.83 Batch B 1.83 Batch C 1.54 Batch D 2.52 Deformulated Control4.34 Unformulated Control 3.35

F. Release Study of Modified mRNA Encapsulated in PLGA Microspheres

PLGA microspheres formulated with Factor IX modified RNA (mRNA sequenceshown in SEQ ID NO: 33901; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) were resuspended in water to a PLGAmicrosphere concentration of 24 mg/ml. After resuspension, 150 ul of thePLGA microsphere suspension was aliquoted into eppendorf tubes. Sampleswere kept incubating and shaking at 37° C. during the course of thestudy. Triplicate samples were pulled at 0.2, 1, 2, 8, 14, and 21 days.To determine the amount of modified RNA released from the PLGAmicrospheres, samples were centrifuged, the supernatant was removed, andthe modified RNA concentration in the supernatant was determined by OD260. The percent release, shown in Table 31, was calculated based on thetotal amount of modified RNA in each sample. After 31 days, 96% of theFactor IX modified RNA was released from the PLGA microsphereformulations.

TABLE 31 Percent Release Time (days) % Release 0 0.0 0.2 27.0 1 37.7 245.3 4 50.9 8 57.0 14 61.8 21 75.5 31 96.4

G. Particle Size Reproducibility of PLGA Microspheres

Three batches of Factor IX modified mRNA (mRNA sequence shown in SEQ IDNO: 33901; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) PLGA microspheres were made using the same conditionsdescribed for Batch D, shown in Table 28, (0.4 ml of W4 at 4 mg/ml, 2.0ml of O1 at 200 mg/ml, 200 ml of W2 at 1%, and homogenized at a speed of5 for the W4/O1/W2 emulsion). To improve the homogeneity of the PLGAmicrosphere suspension, filtration was incorporated prior tocentrifugation. After stirring for 3 hours and before centrifuging, allformulated material was passed through a 100 μm nylon mesh strainer(Fisherbrand Cell Strainer, Cat #22-363-549) to remove largeraggregates. After washing and resuspension with water, 100-200 μl of aPLGA microspheres sample was used to measure particle size of theformulations by laser diffraction (Malvern Mastersizer2000). Theparticle size of the samples is shown in Table 32.

TABLE 32 Particle Size Summary Volume Weighted ID D10 (μm) D50 (μm) D90(μm) Mean (um) Filtration Control 19.2 62.5 722.4 223.1 No A 9.8 31.665.5 35.2 Yes B 10.5 32.3 66.9 36.1 Yes C 10.8 35.7 79.8 41.4 Yes

Results of the 3 PLGA microsphere batches using filtration were comparedto a PLGA microsphere batch made under the same conditions withoutfiltration. The inclusion of a filtration step before washing reducedthe mean particle size and demonstrated a consistent particle sizedistribution between 3 PLGA microsphere batches.

H. Serum Stability of Factor IX PLGA Microspheres

Factor IX mRNA mRNA (mRNA sequence shown in SEQ ID NO: 33901; polyA tailof approximately 160 nucleotides not shown in sequence; 5′cap, Cap1;fully modified with 5-methylcytosine and pseudouridine) in buffer (TE)or 90% serum (Se), or Factor IX mRNA in PLGA in buffer, 90% serum or 1%serum was incubated in buffer, 90% serum or 1% serum at an mRNAconcentration of 50 ng/ul in a total volume of 70 ul. The samples wereremoved at 0, 30, 60 or 120 minutes. RNases were inactivated withproteinase K digestion for 20 minutes at 55° C. by adding 25 ul of 4×proteinase K buffer (0.4 ml 1M TRIS-HCl pH 7.5, 0.1 ml 0.5M EDTA, 0.12ml 5M NaCl, and 0.4 ml 10% SDS) and 8 ul of proteinase K at 20 mg/ml.The Factor IX mRNA was precipitated (add 250 ul 95% ethanol for 1 hour,centrifuge for 10 min at 13 k rpm and remove supernatant, add 200 ul 70%ethanol to the pellet, centrifuge again for 5 min at 13 k rpm and removesupernatant and resuspend the pellet in 70 ul water) or extracted fromPLGA microspheres (centrifuge 5 min at 13 k rpm and remove supernatant,wash pellet with 1 ml water, centrifuge 5 min at 13 k rpm and removesupernatant, add 280 ul dichloromethane to the pellet and shake for 15minutes, add 70 ul water and then shake for 2 hours and remove theaqueous phase) before being analyzed by bioanalyzer. PLGA microspheresprotect Factor IX modified mRNA from degradation in 90% and 1% serumover 2 hours. Factor IX modified mRNA completely degrades in 90% serumat the initial time point.

Example 17 Lipid Nanoparticle In Vivo Studies

G-CSF (cDNA with the T7 promoter, 5′ Untranslated region (UTR) and 3′UTRused in in vitro transcription is given in SEQ ID NO: 33896. mRNAsequence shown in SEQ ID NO: 33897; polyA tail of approximately 160nucleotides not shown in sequence; 5′cap, Cap 1; fully modified with5-methylcytosine and pseudouridine) and Factor IX (cDNA with the T7promoter, 5′ UTR and 3′UTR used in in vitro transcription is given inSEQ ID NO: 33904. mRNA sequence shown in SEQ ID NO: 33901; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1;fully modified with 5-methylcytosine and pseudouridine) modified mRNAwere formulated as lipid nanoparticles (LNPs) using the syringe pumpmethod. The LNPs were formulated at a 20:1 weight ratio of total lipidto modified mRNA with a final lipid molar ratio of 50:10:38.5:1.5(DLin-KC2-DMA:DSPC:Cholesterol:PEG-c-DOMG). Formulations, listed inTable 33, were characterized by particle size, zeta potential, andencapsulation.

TABLE 33 Formulations Formulation # NPA-029-1 NPA-030-1 Modified mRNAFactor IX G-CSF Mean size 91 nm 106 nm PDI: 0.04 PDI: 0.06 Zeta at pH7.4 1.8 mV 0.9 mV Encaps. 92% 100% (RiboGr)

LNP formulations were administered to mice (n=5) intravenously at amodified mRNA dose of 100, 10, or 1 ug. Mice were sacrificed at 8 hrsafter dosing. Serum was collected by cardiac puncture from mice thatwere administered with G-CSF or Factor IX modified mRNA formulations.Protein expression was determined by ELISA.

There was no significant body weight loss (<5%) in the G-CSF or FactorIX dose groups. Protein expression for G-CSF or Factor IX dose groupswas determined by ELISA from a standard curve. Serum samples werediluted (about 20-2500× for G-CSF and about 10-250× for Factor IX) toensure samples were within the linear range of the standard curve. Asshown in Table 34, G-CSF protein expression determined by ELISA wasapproximately 17, 1200, and 4700 ng/ml for the 1, 10, and 100 ug dosegroups, respectively. As shown in Table 35, Factor IX protein expressiondetermined by ELISA was approximately 36, 380, and 3000-11000 ng/ml forthe 1, 10, and 100 ug dose groups, respectively.

TABLE 34 G-CSF Protein Expression Dose (ug) Conc (ng/ml) Dilution FactorSample Volume 1 17.73  20x   5 ul 10 1204.82 2500x 0.04 ul 100 4722.202500x 0.04 ul

TABLE 35 Factor IX Protein Expression Dose (ug) Conc (ng/ml) DilutionFactor Sample Volume  1 36.05  10x   5 ul 10 383.04  10x   5 ul 100*3247.75  50x   1 ul 100* 11177.20 250x 0.2 ul

As shown in Table 36, the LNP formulations described above have about a10,000-100,000-fold increase in protein production compared to anadministration of an intravenous (IV)-lipoplex formulation for the samedosage of modified mRNA and intramuscular (IM) or subcutaneous (SC)administration of the same dose of modified mRNA in saline. As used inTable 36, the symbol “˜” means about.

TABLE 36 Protein Production Serum Concentration (pg/ml) Dose (ug) 8-12hours after administration G-CSF IM 100 ~20-80 SC 100 ~10-40 IV(Lipoplex) 100 ~30 IV (LNP) 100 ~5,000,000 IV (LNP) 10 ~1,000,000 IV(LNP) 1 ~20,000 Factor IX IM 2 × 100 ~1.6 ng/ml IV (LNP) 100~3,000-10,000 ng/ml IV (LNP) 10 ~400 ng/ml IV (LNP) 1 ~40 ng/ml

Materials and Methods for Examples 18-23

G-CSF (mRNA sequence shown in SEQ ID NO: 33897; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap 1; fullymodified with 5-methylcytosine and pseudouridine) and EPO (mRNA sequenceshown in SEQ ID NO: 33900; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′cap, Cap 1; fully modified with5-methylcytosine and pseudouridine) modified mRNA were formulated aslipid nanoparticles (LNPs) using the syringe pump method. The LNPs wereformulated at a 20:1 weight ratio of total lipid to modified mRNA with afinal lipid molar ratio of 50:10:38.5:1.5(DLin-KC2-DMA:DSPC:Cholesterol:PEG-c-DOMG). Formulations, listed inTable 37, were characterized by particle size, zeta potential, andencapsulation.

TABLE 37 Formulations Formulation # NPA-030-2 NPA-060-1 Modified mRNAG-CSF EPO Mean size 84 nm 85 nm PDI: 0.04 PDI: 0.03 Zeta at pH 7.4 0.8mV 1.5 mV Encapsulation 95% 98% (RiboGreen)

Example 18 Lipid Nanoparticle In Vivo Studies with Modified mRNA

LNP formulations, shown in Table 37 (above), were administered to rats(n=5) intravenously (IV), intramuscularly (IM) or subcutaneously (SC) ata single modified mRNA dose of 0.05 mg/kg. A control group of rats (n=4)was untreated. The rats were bled at 2 hours, 8 hours, 24 hours, 48hours and 96 hours and after they were administered with G-CSF or EPOmodified mRNA formulations to determine protein expression using ELISA.The rats administered EPO modified mRNA intravenously were also bled at7 days.

As shown in Table 38, EPO protein expression in the rats intravenouslyadministered modified EPO mRNA was detectable out to 5 days. G-CSF inthe rats intravenously administered modified G-CSF mRNA was detectableto 7 days. Subcutaneous and intramuscular administration of EPO modifiedmRNA was detectable to at least 24 hours and G-CSF modified mRNA wasdetectable to at least 8 hours. In Table 38, “OSC” refers to values thatwere outside the standard curve and “NT” means not tested.

TABLE 38 G-CSF and EPO Protein Expression EPO Serum G-CSF SerumConcentration Concentration Route Time (pg/ml) (pg/ml) IV  2 hours36,981.0 31,331.9 IV  8 hours 62,053.3 70,532.4 IV 24 hours 42,077.05,738.6 IV 48 hours 5,561.5 233.8 IV  5 days 0.0 60.4 IV  7 days 0.0 NTIM  2 hours 1395.4 1620.4 IM  8 hours 8974.6 7910.4 IM 24 hours 4678.3893.3 IM 48 hours NT OSC IM  5 days NT OSC SC  2 hours 386.2 80.3 SC  8hours 985.6 164.2 SC 24 hours 544.2 OSC SC 48 hours NT OSC SC  5 days NTOSC Untreated All bleeds 0 0

Example 19 Time Course In Vivo Study

LNP formulations, shown in 37 (above), were administered to mice (n=5)intravenously (IV) at a single modified mRNA dose of 0.5, 0.05 or 0.005mg/kg. The mice were bled at 8 hours, 24 hours, 72 hours and 6 daysafter they were administered with G-CSF or EPO modified mRNAformulations to determine protein expression using ELISA.

As shown in Table 39, EPO and G-CSF protein expression in the miceadministered with the modified mRNA intravenously was detectable out to72 hours for the mice dosed with 0.005 mg/kg and 0.05 mg/kg of modifiedmRNA and out to 6 days for the mice administered the EPO modified mRNA.In Table 39, “>” means greater than and “ND” means not detected.

TABLE 39 Protein Expression EPO Serum G-CSF Serum Dose ConcentrationConcentration (mg/kg) Time (pg/ml) (pg/ml) 0.005  8 hours 12,508.311,550.6 0.005 24 hours 6,803.0 5,068.9 0.005 72 hours ND ND 0.005  6days ND ND 0.05  8 hours 92,139.9 462,312.5 0.05 24 hours 54,389.480,903.8 0.05 72 hours ND ND 0.05  6 days ND ND 0.5  8 hours498,515.3 >1,250,000 0.5 24 hours 160,566.3 495,812.5 0.5 72 hours3,492.5 1,325.6 0.5  6 days 21.2 ND

Example 20 LNP Formulations In Vivo Study in Rodents

A. LNP Formulations in Mice

LNP formulations, shown in Table 37 (above), were administered to mice(n=4) intravenously (IV) at a single modified mRNA dose 0.05 mg/kg or0.005 mg/kg. There was also 3 control groups of mice (n=4) that wereuntreated. The mice were bled at 2 hours, 8 hours, 24 hours, 48 hoursand 72 hours after they were administered with G-CSF or EPO modifiedmRNA formulations to determine the protein expression. Proteinexpression of G-CSF and EPO were determined using ELISA.

As shown in Table 40, EPO and G-CSF protein expression in the mice wasdetectable at least out to 48 hours for the mice that received a dose of0.005 mg/kg modified RNA and 72 hours for the mice that received a doseof 0.05 mg/kg modified RNA. In Table 40, “OSC” refers to values thatwere outside the standard curve and “NT” means not tested.

TABLE 40 Protein Expression in Mice EPO Serum G-CSF Serum DoseConcentration Concentration (mg/kg) Time (pg/ml) (pg/ml) 0.005  2 hoursOSC 3,447.8 0.005  8 hours 1,632.8 11,454.0 0.005 24 hours 1,141.04,960.2 0.005 48 hours 137.4 686.4 0.005 72 hours 0 NT 0.05  2 hours10,027.3 20,951.4 0.05  8 hours 56,547.2 70,012.8 0.05 24 hours 25,027.319,356.2 0.05 48 hours 1,432.3 1,963.0 0.05 72 hours 82.2 47.3

B. LNP Formulations in Rats

LNP formulations, shown in Table 37 (above), are administered to rats(n=4) intravenously (IV) at a single modified mRNA dose 0.05 mg/kg.There is also a control group of rats (n=4) that are untreated. The ratsare bled at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, 7 days and14 days after they were administered with G-CSF or EPO modified mRNAformulations to determine the protein expression. Protein expression ofG-CSF and EPO are determined using ELISA.

Example 21 Early Time Course Study of LNPs

LNP formulations, shown in Table 37 (above), are administered to mammalsintravenously (IV), intramuscularly (IM) or subcutaneously (SC) at asingle modified mRNA dose of 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg. Acontrol group of mammals are not treated. The mammals are bled at 5minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5hours and/or 2 hours after they are administered with the modified mRNALNP formulations to determine protein expression using ELISA. Themammals are also bled to determine the complete blood count such as thegranulocyte levels and red blood cell count.

Example 22 Non-Human Primate In Vivo Study

LNP formulations, shown in Table 37 (above), were administered tonon-human primates (NHP) (cynomolgus monkey) (n=2) as a bolusintravenous injection (IV) over approximately 30 seconds using ahypodermic needle, which may be attached to a syringe/abbocath orbutterfly if needed. The NHP were administered a single modified mRNA IVdose of 0.05 mg/kg of EPO or G-CSF or 0.005 mg/kg of EPO in a dosevolume of 0.5 mL/kg. The NHPs were bled 5-6 days before dosing with themodified mRNA LNP formulations to determine protein expression in theserum and a baseline complete blood count. After administration with themodified mRNA formulation the NHP were bled at 8, 24, 48 and 72 hours todetermined protein expression. At 24 and 72 hours after administrationthe complete blood count of the NHP was also determined. Proteinexpression of G-CSF and EPO was determined by ELISA. Urine from the NHPswas collected over the course of the entire experiment and analyzed toevaluate clinical safety. Samples were collected from the NHPs afterthey were administered with G-CSF or EPO modified mRNA formulations todetermine protein expression using ELISA. Clinical chemistry,hematology, urinalysis and cytokines of the non-human primates were alsoanalyzed.

As shown in Table 41, EPO protein expression in the NHPs administered0.05 mg/kg is detectable out to 72 hours and the 0.005 mg/kg dosing ofthe EPO formulation is detectable out to 48 hours. In Table 41, the “<”means less than a given value. G-CSF protein expression was seen out to24 hours after administration with the modified mRNA formulation.Preliminarily, there was an increase in granulocytes and reticulocyteslevels seen in the NHP after administration with the modified mRNAformulations.

TABLE 41 Protein Expression in Non-Human Primates Female NHP Male NHPAverage Serum Serum Serum Concen- Concen- Conen- Modified Dose trationtration tration mRNA (mg/kg) Time (pg/ml) (pg/ml) (pg/ml) G-CSF 0.05Pre-bleed 0 0 0  8 hours 3289 1722 2,506 24 hours 722 307 515 48 hours 00 0 72 hours 0 0 0 EPO 0.05 Pre-bleed 0 0 0  8 hours 19,858 7,072 13,46524 hours 18,178 4,913 11,546 48 hours 5,291 498 2,895 72 hours 744 60402 EPO 0.005 Pre-bleed 0 0 0  8 hours 523 250 387 24 hours 302 113 20848 hours <7.8 <7.8 <7.8 72 hours 0 0 0

Example 23 Non-Human Primate In Vivo Study for G-CSF and EPO

LNP formulations, shown in Table 37 (above), were administered tonon-human primates (NHP) (cynomolgus monkey) (n=2) as intravenousinjection (IV). The NHP were administered a single modified mRNA IV doseof 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg of G-CSF or EPO in a dose volumeof 0.5 mL/kg. The NHPs were bled before dosing with the modified mRNALNP formulations to determine protein expression in the serum and abaseline complete blood count. After administration with the G-CSFmodified mRNA formulation the NHP were bled at 8, 24, 48 and 72 hours todetermined protein expression. After administration with the EPOmodified mRNA formulation the NHP were bled at 8, 24, 48, 72 hours and 7days to determined protein expression.

Samples collected from the NHPs after they were administered with G-CSFor EPO modified mRNA formulations were analyzed by ELISA to determineprotein expression. Neutrophil and reticulocyte count was alsodetermined pre-dose, 24 hours, 3 days, 7 days, 14 days and 18 days afteradministration of the modified G-CSF or EPO formulation.

As shown in Table 42, G-CSF protein expression was not detected beyond72 hours. In Table 42, “<39” refers to a value below the lower limit ofdetection of 39 pg/ml.

TABLE 42 G-CSF Protein Expression Female NHP Male NHP Serum Serum G-CSFG-CSF Concen- Concen- Modified Dose tration tration mRNA (mg/kg) Time(pg/ml) (pg/ml) G-CSF 0.5 Pre-bleed <39 <39  8 hours 43,525 43,594 24hours 11,374 3,628 48 hours 1,100 833 72 hours <39 306 G-CSF 0.05Pre-bleed <39 <39  8 hours 3,289 1,722 24 hours 722 307 48 hours <39 <3972 hours <39 <39 G-CSF 0.005 Pre-bleed <39 <39  8 hours 559 700 24 hours155 <39 48 hours <39 <39 72 hours <39 <39

As shown in Table 43, EPO protein expression was not detected beyond 7days. In Table 43, “<7.8” refers to a value below the lower limit ofdetection of 7.8 pg/ml.

TABLE 43 EPO Protein Expression Female NHP Male NHP Serum Serum EPO EPOConcen- Concen- Modified Dose tration tration mRNA (mg/kg) Time (pg/ml)(pg/ml) EPO 0.5 Pre-bleed <7.8 <7.8  8 hours 158,771 119,086 24 hours133,978 85,825 48 hours 45,250 64,793 72 hours 15,097 20,407  7 days<7.8 <7.8 EPO 0.05 Pre-bleed <7.8 <7.8  8 hours 19,858 7,072 24 hours18,187 4,913 48 hours 5,291 498 72 hours 744 60  7 days <7.8 <7.8 EPO0.005 Pre-bleed <7.8 <7.8  8 hours 523 250 24 hours 302 113 48 hours 1129 72 hours <7.8 <7.8  7 days <7.8 <7.8

As shown in Table 44, there was an increase in neutrophils in all G-CSFgroups relative to pre-dose levels.

TABLE 44 Pharmacologic Effect of G-CSF mRNA in NHP Male NHP Female MaleNHP Female (G-CSF) NHP (EPO) NHP Neutro- (G-CSF) Neutro- (EPO) Dosephils Neutrophils phils Neutrophils (mg/kg) Time (10⁹/L) (10⁹/L) (10⁹/L)(10⁹/L) 0.5  Pre-dose  1.53 1.27 9.72 1.82 24 hours 14.92 13.96 7.5 11.85  3 days  9.76 13.7 11.07  5.22 7 days  2.74 3.81 11.8  2.85 14/18days  2.58 1.98 7.16 2.36 0.05  Pre-dose 13.74 3.05 0.97 2.15 24 hours19.92 29.91 2.51 2.63 3 days  7.49 10.77 1.73 4.08 7 days  4.13 3.8 1.232.77 14/18 days  3.59 1.82 1.53 1.27 0.005 Pre-dose  1.52 2.54 5.46 5.9624 hours 16.44 8.6 5.37 2.59 3 days  3.74 1.78 6.08 2.83 7 days  7.282.27 3.51 2.23 14/18 days  4.31 2.28 1.52 2.54

As shown in Table 45, there was an increase in reticulocytes in all EPOgroups 3 days to 14/18 days after dosing relative to reticulocyte levels24 hours after dosing.

TABLE 45 Pharmacologic Effect of EPO mRNA on Neutrophil Count Male NHPFemale Male NHP Female (G-CSF) NHP (EPO) NHP Neutro- (G-CSF) Neutro-(EPO) Dose phils Neutrophils phils Neutrophils (mg/kg) Time (10¹²/L)(10¹²/L) (10¹²/L) (10¹²/L) 0.5  Pre-dose 0.067 0.055 0.107 0.06  24hours 0.032 0.046 0.049 0.045 3 days 0.041 0.017 0.09  0.064 7 days0.009 0.021 0.35  0.367 14/18 days 0.029 0.071 0.066 0.071 0.05 Pre-dose 0.055 0.049 0.054 0.032 24 hours 0.048 0.046 0.071 0.04  3 days0.101 0.061 0.102 0.105 7 days 0.157 0.094 0.15  0.241 14/18 days 0.1070.06  0.067 0.055 0.005 Pre-dose 0.037 0.06  0.036 0.052 24 hours 0.0370.07  0.034 0.061 3 days 0.037 0.054 0.079 0.118 7 days 0.046 0.0660.049 0.087 14/18 days 0.069 0.057 0.037 0.06 

As shown in Tables 46-48, the administration of EPO modified RNA had aneffect on other erythropoietic parameters including hemoglobin (HGB),hematocrit (HCT) and red blood cell (RBC) count.

TABLE 46 Pharmacologic Effect of EPO mRNA on Hemoglobin Male NHP FemaleNHP Male NHP Female NHP (G-CSF) (G-CSF) (EPO) (EPO) Dose HGB HGB HGB HGB(mg/kg) Time (g/L) (g/L) (g/L) (g/L) 0.5  Pre-dose 133 129 134 123 24hours 113 112 127 108 3 days 118 114 126 120 7 days 115 116 140 13414/18 days 98 113 146 133 0.05  Pre-dose 137 129 133 133 24 hours 122117 123 116 3 days 126 115 116 120 7 days 126 116 126 121 14/18 days 134123 133 129 0.005 Pre-dose 128 129 132 136 24 hours 117 127 122 128 3days 116 127 125 130 7 days 116 129 119 127 14/18 days 118 129 128 129

TABLE 47 Pharmacologic Effect of EPO mRNA on Hematocrit Male NHP FemaleNHP Male NHP Female NHP (G-CSF) (G-CSF) (EPO) (EPO) Dose HCT HCT HCT HCT(mg/kg) Time (L/L) (L/L) (L/L) (L/L) 0.5  Pre-dose 0.46 0.43 0.44 0.4 24hours 0.37 0.38 0.4 0.36 3 days 0.39 0.38 0.41 0.39 7 days 0.39 0.380.45 0.45 14/18 days 0.34 0.37 0.48 0.46 0.05  Pre-dose 0.44 0.44 0.450.43 24 hours 0.39 0.4 0.43 0.39 3 days 0.41 0.39 0.38 0.4 7 days 0.420.4 0.45 0.41 14/18 days 0.44 0.4 0.46 0.43 0.005 Pre-dose 0.42 0.420.48 0.45 24 hours 0.4 0.42 0.42 0.43 3 days 0.4 0.41 0.44 0.42 7 days0.39 0.42 0.41 0.42 14/18 days 0.41 0.42 0.42 0.42

TABLE 48 Pharmacologic Effect of EPO mRNA on Red Blood Cells Male NHP(G- Female NHP (G- Female NHP Dose CSF) RBC CSF) RBC Male NHP (EPO)(EPO) RBC (mg/kg) Time (10¹²/L) (10¹²/L) RBC (10¹²/L) (10¹²/L) 0.5Pre-dose 5.57 5.57 5.43 5.26 24 hours 4.66 4.96 5.12 4.69 3 days 4.914.97 5.13 5.15 7 days 4.8 5.04 5.55 5.68 14/18 days 4.21 4.92 5.83 5.720.05 Pre-dose 5.68 5.64 5.57 5.84 24 hours 4.96 5.08 5.25 5.18 3 days5.13 5.04 4.81 5.16 7 days 5.17 5.05 5.37 5.31 14/18 days 5.43 5.26 5.575.57 0.005 Pre-dose 5.67 5.36 6.15 5.72 24 hours 5.34 5.35 5.63 5.35 3days 5.32 5.24 5.77 5.42 7 days 5.25 5.34 5.49 5.35 14/18 days 5.37 5.345.67 5.36

As shown in Tables 49 and 50, the administration of modified RNA had aneffect on serum chemistry parameters including alanine transaminase(ALT) and aspartate transaminase (AST).

TABLE 49 Pharmacologic Effect of EPO mRNA on Alanine Transaminase FemaleNHP Dose Male NHP (G- Female NHP (G- Male NHP (EPO) (EPO) ALT (mg/kg)Time CSF) ALT (U/L) CSF) ALT (U/L) ALT (U/L) (U/L) 0.5 Pre-dose 29 21650 31  2 days 63 209 98 77  4 days 70 98 94 87  7 days 41 149 60 59 14days 43 145 88 44 0.05 Pre-dose 58 53 56 160  2 days 82 39 95 254  4days 88 56 70 200  7 days 73 73 64 187 14 days 50 31 29 216 0.005Pre-dose 43 51 45 45  2 days 39 32 62 48  4 days 48 58 48 50  7 days 2955 21 48 14 days 44 46 43 51

TABLE 50 Pharmacologic Effect of EPO mRNA on Aspartate Transaminase DoseMale NHP (G- Female NHP (G- Male NHP (EPO) Female NHP (mg/kg) Time CSF)AST (U/L) CSF) AST (U/L) AST (U/L) (EPO) AST (U/L) 0.5 Pre-dose 32 47 5920  2 days 196 294 125 141  4 days 67 63 71 60  7 days 53 68 56 47 14days 47 67 82 44 0.05 Pre-dose 99 33 74 58  2 days 95 34 61 80  4 days69 42 48 94  7 days 62 52 53 78 14 days 59 20 32 47 0.005 Pre-dose 35 5439 40  2 days 70 34 29 25  4 days 39 36 43 55  7 days 28 31 55 31 14days 39 20 35 54

As shown in Table 51, the administration of lipidnanoparticle-formulated modified RNA at high doses (0.5 mg/kg) caused anincrease in cytokines, interferon-alpha (IFN-alpha) after administrationof modified mRNA.

TABLE 51 Pharmacologic Effect of EPO mRNA on Alanine Transaminase MaleNHP (G- Female NHP (G- Male NHP (EPO) Female NHP Dose CSF) IFN-alphaCSF) IFN-alpha IFN-alpha (EPO) IFN-alpha (mg/kg) Time (pg/mL) (pg/mL)(pg/mL) (pg/mL) 0.5 Pre-dose 0 0 0 0 Day 1 + 8 hr 503.8 529.2 16.79217.5 4 days 0 0 0 0 0.05 Pre-dose 0 0 0 0 Day 1 + 8 hr 0 0 0 0 4 days 00 0 0 0.005 Pre-dose 0 0 0 0 Day 1 + 8 hr 0 0 0 0 4 days 0 0 0 0

Example 24 Study of Intramuscular and/or Subcutaneous Administration inNon-Human Primates

Formulations containing modified EPO mRNA (mRNA sequence shown in SEQ IDNO: 33900; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) or G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 33897;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) insaline were administered to non-human primates (Cynomolgus monkey) (NHP)intramuscularly (IM) or subcutaneously (SC). The single modified mRNAdose of 0.05 mg/kg or 0.005 mg/kg was in a dose volume of 0.5 mL/kg. Thenon-human primates are bled 5-6 days prior to dosing to determine serumprotein concentration and a baseline complete blood count. Afteradministration with the modified mRNA formulation the NHP are bled at 8hours, 24 hours, 48 hours, 72 hours, 7 days and 14 days to determinedprotein expression. Protein expression of G-CSF and EPO is determined byELISA. At 24 hours, 72 hours, 7 days and 14 days after administrationthe complete blood count of the NHP is also determined. Urine from theNHPs is collected over the course of the entire experiment and analyzedto evaluate clinical safety. Tissue near the injection site is alsocollected and analyzed to determine protein expression.

Example 25 Modified mRNA Trafficking

In order to determine localization and/or trafficking of the modifiedmRNA, studies may be performed as follows.

LNP formulations of siRNA and modified mRNA are formulated according tomethods known in the art and/or described herein. The LNP formulationsmay include at least one modified mRNA which may encode a protein suchas G-CSF, EPO, Factor VII, and/or any protein described herein. Theformulations may be administered locally into muscle of mammals usingintramuscular or subcutaneous injection. The dose of modified mRNA andthe size of the LNP may be varied to determine the effect on traffickingin the body of the mammal and/or to assess the impact on a biologicreaction such as, but not limited to, inflammation. The mammal may bebled at different time points to determine the expression of proteinencoded by the modified mRNA administered present in the serum and/or todetermine the complete blood count in the mammal.

For example, modified mRNA encoding Factor VII, expressed in the liverand secreted into the serum, may be administered intramuscularly and/orsubcutaneously. Coincident or prior to modified mRNA administration,siRNA is administered to knock out endogenous Factor VII. Factor VIIarising from the intramuscular and/or subcutaneous injection of modifiedmRNA is administered is measured in the blood. Also, the levels ofFactor VII is measured in the tissues near the injection site. If FactorVII is expressed in blood then there is trafficking of the modifiedmRNA. If Factor VII is expressed in tissue and not in the blood thanthere is only local expression of Factor VII.

Example 26 Formulations of Multiple Modified mRNA

LNP formulations of modified mRNA are formulated according to methodsknown in the art and/or described herein or known in the art. The LNPformulations may include at least one modified mRNA which may encode aprotein such as G-CSF, EPO, thrombopoietin and/or any protein describedherein. The at least one modified mRNA may include 1, 2, 3, 4 or 5modified mRNA molecules. The formulations containing at least onemodified mRNA may be administered intravenously, intramuscularly orsubcutaneously in a single or multiple dosing regimens. Biologicalsamples such as, but not limited to, blood and/or serum may be collectedand analyzed at different time points before and/or after administrationof the at least one modified mRNA formulation. An expression of aprotein in a biological sample of 50-200 pg/ml after the mammal has beenadministered a formulation containing at least one modified mRNAencoding said protein would be considered biologically effective.

Example 27 Polyethylene Glycol Ratio Studies

A. Formulation and Characterization of PEG LNPs

Lipid nanoparticles (LNPs) were formulated using the syringe pumpmethod. The LNPs were formulated at a 20:1 weight ratio of total lipidto modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 33897; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and pseudouridine). The molarratio ranges of the formulations are shown in Table 52.

TABLE 52 Molar Ratios DLin-KC2-DMA DSPC Cholesterol PEG-c-DOMG MolePercent 50.0 10.0 37-38.5 1.5-3 (mol %)

Two types of PEG lipid, 1,2-Dimyristoyl-sn-glycerol, methoxypolyethyleneGlycol (PEG-DMG, NOF Cat #SUNBRIGHT® GM-020) and1,2-Distearoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DSG, NOF Cat#SUNBRIGHT® GS-020), were tested at 1.5 or 3.0 mol %. After theformation of the LNPs and the encapsulation of the modified G-CSF mRNA,the LNP formulations were characterized by particle size, zeta potentialand encapsulation percentage and the results are shown in Table 53.

TABLE 53 Characterization of LNP Formulations Formulation No. NPA-071-1NPA-072-1 NPA-073-1 NPA-074-1 Lipid PEG-DMG PEG-DMG PEG-DSA PEG-DSA 1.5%3% 1.5% 3% Mean Size 95 nm 85 nm 95 nm 75 nm PDI: 0.01 PDI: 0.06 PDI:0.08 PDI: 0.08 Zeta at pH 7.4 −1.1 mV −2.6 mV 1.7 mV 0.7 mVEncapsulation 88% 89% 98% 95% (RiboGreen)

B. In Vivo Screening of PEG LNPs

Formulations of the PEG LNPs described in Table 54 were administered tomice (n=5) intravenously at a dose of 0.5 mg/kg. Serum was collectedfrom the mice at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours and 8days after administration of the formulation. The serum was analyzed byELISA to determine the protein expression of G-CSF and the expressionlevels are shown in Table 54. LNP formulations using PEG-DMG gavesubstantially higher levels of protein expression than LNP formulationswith PEG-DSA.

TABLE 54 Protein Expression Formulation Protein Expression Lipid No.Time (pg/ml) PEG-DMG, NPA-071-1  2 hours 114,102 1.5%  8 hours 357,94424 hours 104,832 48 hours 6,697 72 hours 980 8 days 0 PEG-DMG, 3%NPA-072-1  2 hours 154,079  8 hours 354,994 24 hours 164,311 48 hours13,048 72 hours 1,182 8 days 13 PEG-DSA, 1.5% NPA-073-1  2 hours 3,193 8 hours 6,162 24 hours 446 48 hours 197 72 hours 124 8 days 5 PEG-DSA,3% NPA-074-1  2 hours 259  8 hours 567 24 hours 258 48 hours 160 72hours 328 8 days 33

Example 28 Cationic Lipid Formulation Studies

A. Formulation and Characterization of Cationic Lipid Nanoparticles

Lipid nanoparticles (LNPs) were formulated using the syringe pumpmethod. The LNPs were formulated at a 20:1 weight ratio of total lipidto modified mRNA. The final lipid molar ratio ranges of cationic lipid,DSPC, cholesterol and PEG-c-DOMG are outlined in Table 55.

TABLE 55 Molar Ratios Cationic Lipid DSPC Cholesterol PEG-c-DOMG MolePercent 50.0 10.0 38.5 1.5 (mol %)

A 25 mM lipid solution in ethanol and modified RNA in 50 mM citrate at apH of 3 were mixed to create spontaneous vesicle formation. The vesicleswere stabilized in ethanol before the ethanol was removed and there wasa buffer exchange by dialysis. The LNPs were then characterized byparticle size, zeta potential, and encapsulation percentage. Table 56describes the characterization of LNPs encapsulating EPO modified mRNA(mRNA sequence shown in SEQ ID NO: 33900 polyA tail of approximately 160nucleotides not shown in sequence; 5′cap, Cap1; fully modified with5-methylcytosine and pseudouridine) or G-CSF modified mRNA (mRNAsequence shown in SEQ ID NO: 33897; polyA tail of approximately 160nucleotides not shown in sequence; 5′cap, Cap1; fully modified with5-methylcytosine and pseudouridine) using DLin-MC3-DMA, DLin-DMA orC12-200 as the cationic lipid.

TABLE 56 Characterization of Cationic Lipid Formulations FormulationNPA-071- NPA-072- NPA-073- NPA-074- NPA-075- NPA-076- No. 1 1 1 1 1 1Lipid DLin- DLin- DLin- DLin- C12-200 C12-200 MC3- MC3- DMA DMA DMA DMAModified EPO G-CSF EPO G-CSF EPO G-CSF RNA Mean Size 89 nm 96 nm 70 nm73 nm 97 nm 103 nm PDI: 0.07 PDI: 0.08 PDI: 0.04 PDI: 0.06 PDI: 0.05PDI: 0.09 Zeta at pH 7.4 −1.1 mV −1.4 mV −1.6 mV −0.4 mV 1.4 mV 0.9 mVEncapsulation 100% 100% 99% 100% 88% 98% (RiboGreen)

B. In Vivo Screening of Cationic LNP Formulations

Formulations of the cationic lipid formulations described in Table 56were administered to mice (n=5) intravenously at a dose of 0.5 mg/kg.Serum was collected from the mice at 2 hours, 24 hours, 72 hours and/or7 days after administration of the formulation. The serum was analyzedby ELISA to determine the protein expression of EPO or G-CSF and theexpression levels are shown in Table 57.

TABLE 57 Protein Expression Protein Modified Expression mRNA FormulationNo. Time (pg/ml) EPO NPA-071-1  2 hours 304,190.0 24 hours 166,811.5 72hours 1,356.1 7 days 20.3 EPO NPA-073-1  2 hours 73,852.0 24 hours75,559.7 72 hours 130.8 EPO NPA-075-1  2 hours 413,010.2 24 hours56,463.8 G-CSF NPA-072-1  2 hours 62,113.1 24 hours 53,206.6 G-CSFNPA-074-1 24 hours 25,059.3 G-CSF NPA-076-1  2 hours 219,198.1 24 hours8,470.0

Toxicity was seen in the mice administered the LNPs formulations withthe cationic lipid C12-200 (NPA-075-1 and NPA-076-1) and they weresacrificed at 24 hours because they showed symptoms such as scrubby fur,cowering behavior and weight loss of greater than 10%. C12-200 wasexpected to be more toxic but also had a high level of expression over ashort period. The cationic lipid DLin-DMA (NPA-073-1 and NPA-074-1) hadthe lowest expression out of the three cationic lipids tested.DLin-MC3-DMA (NPA-071-1 and NPA-072-1) showed good expression up to daythree and was above the background sample out to day 7 for EPOformulations.

Example 29 Method of Screening for Protein Expression

A. Electrospray Ionization

A biological sample which may contain proteins encoded by modified RNAadministered to the subject is prepared and analyzed according to themanufacturer protocol for electrospray ionization (ESI) using 1, 2, 3 or4 mass analyzers. A biologic sample may also be analyzed using a tandemESI mass spectrometry system.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

B. Matrix-Assisted Laser Desorption/Ionization

A biological sample which may contain proteins encoded by modified RNAadministered to the subject is prepared and analyzed according to themanufacturer protocol for matrix-assisted laser desorption/ionization(MALDI).

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry

A biological sample, which may contain proteins encoded by modified RNA,may be treated with a trypsin enzyme to digest the proteins containedwithin. The resulting peptides are analyzed by liquidchromatography-mass spectrometry-mass spectrometry (LC/MS/MS). Thepeptides are fragmented in the mass spectrometer to yield diagnosticpatterns that can be matched to protein sequence databases via computeralgorithms. The digested sample may be diluted to achieve 1 ng or lessstarting material for a given protein. Biological samples containing asimple buffer background (e.g. water or volatile salts) are amenable todirect in-solution digest; more complex backgrounds (e.g. detergent,non-volatile salts, glycerol) require an additional clean-up step tofacilitate the sample analysis.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

Example 30 Lipid Nanoparticle In Vivo Studies

mCherry mRNA (mRNA sequence shown in SEQ ID NO: 33905; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) was formulated as alipid nanoparticle (LNP) using the syringe pump method. The LNP wasformulated at a 20:1 weight ratio of total lipid to modified mRNA with afinal lipid molar ratio of 50:10:38.5:1.5(DLin-KC2-DMA:DSPC:Cholesterol:PEG-c-DOMG). The mCherry formulation,listed in Table 58, was characterized by particle size, zeta potential,and encapsulation.

TABLE 58 mCherry Formulation Formulation # NPA-003-5 Modified mRNAmCherry Mean size 105 nm PDI: 0.09 Zeta at pH 7.4 1.8 mV Encaps. 100%(RiboGr)

The LNP formulation was administered to mice (n=5) intravenously at amodified mRNA dose of 100 ug. Mice were sacrificed at 24 hrs afterdosing. The liver and spleen from the mice administered with mCherrymodified mRNA formulations were analyzed by immunohistochemistry (IHC),western blot, or fluorescence-activated cell sorting (FACS).

Histology of the liver showed uniform mCherry expression throughout thesection, while untreated animals did not express mCherry. Western blotswere also used to confirm mCherry expression in the treated animals,whereas mCherry was not detected in the untreated animals. Tubulin wasused as a control marker and was detected in both treated and untreatedmice, indicating that normal protein expression in hepatocytes wasunaffected.

FACS and IHC were also performed on the spleens of mCherry and untreatedmice. All leukocyte cell populations were negative for mCherryexpression by FACS analysis. By IHC, there were also no observabledifferences in the spleen in the spleen between mCherry treated anduntreated mice.

Example 31 Syringe Pump In Vivo Studies

mCherry modified mRNA (mRNA sequence shown in SEQ ID NO: 33898; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1) is formulated as a lipid nanoparticle (LNP) using the syringe pumpmethod. The LNP is formulated at a 20:1 weight ratio of total lipid tomodified mRNA with a final lipid molar ratio of 50:10:38.5:1.5(DLin-KC2-DMA:DSPC:Cholesterol:PEG-c-DOMG). The mCherry formulation ischaracterized by particle size, zeta potential, and encapsulation.

The LNP formulation is administered to mice (n=5) intravenously at amodified mRNA dose of 10 or 100 ug. Mice are sacrificed at 24 hrs afterdosing. The liver and spleen from the mice administered with mCherrymodified mRNA formulations are analyzed by immunohistochemistry (IHC),western blot, and/or fluorescence-activated cell sorting (FACS).

Example 32 In Vitro and In Vivo Expression

A. In vitro Expression in Human Cells Using Lipidoid Formulations

The ratio of mmRNA to lipidoid used to test for in vitro transfection istested empirically at different lipidoid:mmRNA ratios. Previous workusing siRNA and lipidoids have utilized 2.5:1, 5:1, 10:1, and 15:1lipidoid:siRNA wt:wt ratios. Given the longer length of mmRNA relativeto siRNA, a lower wt:wt ratio of lipidoid to mmRNA may be effective. Inaddition, for comparison mmRNA were also formulated using RNAIMAX™(Invitrogen, Carlsbad, Calif.) or TRANSIT-mRNA (Mirus Bio, Madison,Wis.) cationic lipid delivery vehicles.

The ability of lipidoid-formulated Luciferase (IVT cDNA sequence asshown in SEQ ID NO: 33906; mRNA sequence shown in SEQ ID NO: 33907,polyA tail of approximately 160 nucleotides not shown in sequence,5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site), green fluorescentprotein (GFP) (IVT cDNA sequence as shown in SEQ ID NO: 3308; mRNAsequence shown in SEQ ID NO: 33909, polyA tail of approximately 160nucleotides not shown in sequence, 5′cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site), G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 33898;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1), and EPO mRNA (mRNA sequence shown in SEQ ID NO: 33900;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1) to express the desired protein product can be confirmed byluminescence for luciferase expression, flow cytometry for GFPexpression, and by ELISA for G-CSF and Erythropoietin (EPO) secretion.

B. In Vivo Expression Following Intravenous Injection

Systemic intravenous administration of the formulations are createdusing various different lipidoids including, but not limited to,98N12-5, C12-200, and MD1.

Lipidoid formulations containing mmRNA are injected intravenously intoanimals. The expression of the modified mRNA (mmRNA)-encoded proteinsare assessed in blood and/or other organs samples such as, but notlimited to, the liver and spleen collected from the animal. Conductingsingle dose intravenous studies will also allow an assessment of themagnitude, dose responsiveness, and longevity of expression of thedesired product.

In one embodiment, lipidoid based formulations of 98N12-5, C12-200, MD1and other lipidoids, are used to deliver luciferase, green fluorescentprotein (GFP), mCherry fluorescent protein, secreted alkalinephosphatase (sAP), human G-CSF, human Factor IX, or human Erythropoietin(EPO) mmRNA into the animal. After formulating mmRNA with a lipid, asdescribed previously, animals are divided into groups to receive eithera saline formulation, or a lipidoid-formulation which contains one of adifferent mmRNA selected from luciferase, GFP, mCherry, sAP, humanG-CSF, human Factor IX, and human EPO. Prior to injection into theanimal, mmRNA-containing lipidoid formulations are diluted in PBS.Animals are then administered a single dose of formulated mmRNA rangingfrom a dose of 10 mg/kg to doses as low as 1 ng/kg, with a preferredrange to be 10 mg/kg to 100 ng/kg, where the dose of mmRNA depends onthe animal body weight such as a 20 gram mouse receiving a maximumformulation of 0.2 ml (dosing is based no mmRNA per kg body weight).After the administration of the mmRNA-lipidoid formulation, serum,tissues, and/or tissue lysates are obtained and the level of themmRNA-encoded product is determined at a single and/or a range of timeintervals. The ability of lipidoid-formulated Luciferase, GFP, mCherry,sAP, G-CSF, Factor IX, and EPO mmRNA to express the desired proteinproduct is confirmed by luminescence for the expression of Luciferase,flow cytometry for the expression of GFP and mCherry expression, byenzymatic activity for sAP, or by ELISA for the section of G-CSF, FactorIX and/or EPO.

Further studies for a multi-dose regimen are also performed to determinethe maximal expression of mmRNA, to evaluate the saturability of themmRNA-driven expression (by giving a control and active mmRNAformulation in parallel or in sequence), and to determine thefeasibility of repeat drug administration (by giving mmRNA in dosesseparated by weeks or months and then determining whether expressionlevel is affected by factors such as immunogenicity). An assessment ofthe physiological function of proteins such as G-CSF and EPO are alsodetermined through analyzing samples from the animal tested anddetecting increases in granulocyte and red blood cell counts,respectively. Activity of an expressed protein product such as FactorIX, in animals can also be assessed through analysis of Factor IXenzymatic activity (such as an activated partial thromboplastin timeassay) and effect of clotting times.

C. In Vitro Expression Following Intramuscular and/or SubcutaneousInjection

The use of lipidoid formulations to deliver oligonucleotides, includingmRNA, via an intramuscular route or a subcutaneous route of injectionneeds to be evaluated as it has not been previously reported.Intramuscular and/or subcutaneous injection of mmRNA are evaluated todetermine if mmRNA-containing lipidoid formulations are capable toproduce both localized and systemic expression of a desired proteins.

Lipidoid formulations of 98N12-5, C12-200, and MD1 containing mmRNAselected from luciferase, green fluorescent protein (GFP), mCherryfluorescent protein, secreted alkaline phosphatase (sAP), human G-CSF,human factor IX, or human Erythropoietin (EPO) mmRNA are injectedintramuscularly and/or subcutaneously into animals. The expression ofmmRNA-encoded proteins are assessed both within the muscle orsubcutaneous tissue and systemically in blood and other organs such asthe liver and spleen. Single dose studies allow an assessment of themagnitude, dose responsiveness, and longevity of expression of thedesired product.

Animals are divided into groups to receive either a saline formulationor a formulation containing modified mRNA. Prior to injectionmmRNA-containing lipidoid formulations are diluted in PBS. Animals areadministered a single intramuscular dose of formulated mmRNA rangingfrom 50 mg/kg to doses as low as 1 ng/kg with a preferred range to be 10mg/kg to 100 ng/kg. A maximum dose for intramuscular administration, fora mouse, is roughly 1 mg mmRNA or as low as 0.02 ng mmRNA for anintramuscular injection into the hind limb of the mouse. Forsubcutaneous administration, the animals are administered a singlesubcutaneous dose of formulated mmRNA ranging from 400 mg/kg to doses aslow as 1 ng/kg with a preferred range to be 80 mg/kg to 100 ng/kg. Amaximum dose for subcutaneous administration, for a mouse, is roughly 8mg mmRNA or as low as 0.02 ng mmRNA.

For a 20 gram mouse the volume of a single intramuscular injection ismaximally 0.025 ml and a single subcutaneous injection is maximally 0.2ml. The optimal dose of mmRNA administered is calculated from the bodyweight of the animal. At various points in time points following theadministration of the mmRNA-lipidoid, serum, tissues, and tissue lysatesis obtained and the level of the mmRNA-encoded product is determined.The ability of lipidoid-formulated luciferase, green fluorescent protein(GFP), mCherry fluorescent protein, secreted alkaline phosphatase (sAP),human G-CSF, human factor IX, or human Erythropoietin (EPO) mmRNA toexpress the desired protein product is confirmed by luminescence forluciferase expression, flow cytometry for GFP and mCherry expression, byenzymatic activity for sAP, and by ELISA for G-CSF, Factor IX andErythropoietin (EPO) secretion.

Additional studies for a multi-dose regimen are also performed todetermine the maximal expression using mmRNA, to evaluate thesaturability of the mmRNA-driven expression (achieved by giving acontrol and active mmRNA formulation in parallel or in sequence), and todetermine the feasibility of repeat drug administration (by giving mmRNAin doses separated by weeks or months and then determining whetherexpression level is affected by factors such as immunogenicity). Studiesutilizing multiple subcutaneous or intramuscular injection sites at onetime point, are also utilized to further increase mmRNA drug exposureand improve protein production. An assessment of the physiologicalfunction of proteins, such as GFP, mCherry, sAP, human G-CSF, humanfactor IX, and human EPO, are determined through analyzing samples fromthe tested animals and detecting a change in granulocyte and/or redblood cell counts. Activity of an expressed protein product such asFactor IX, in animals can also be assessed through analysis of Factor IXenzymatic activity (such as an activated partial thromboplastin timeassay) and effect of clotting times.

Example 33 Bifunctional mmRNA

Using the teachings and synthesis methods described herein, modifiedRNAs are designed and synthesized to be bifunctional, thereby encodingone or more cytotoxic protein molecules as well as be synthesized usingcytotoxic nucleosides.

Administration of the bifunctional modified mRNAs is effected usingeither saline or a lipid carrier. Once administered, the bifunctionalmodified mRNA is translated to produce the encoded cytotoxic peptide.Upon degradation of the delivered modified mRNA, the cytotoxicnucleosides are released which also effect therapeutic benefit to thesubject.

Example 34 Modified mRNA Transfection

A. Reverse Transfection

For experiments performed in a 24-well collagen-coated tissue cultureplate, Keratinocytes are seeded at a cell density of 1×10⁵. Forexperiments performed in a 96-well collagen-coated tissue culture plate,Keratinocytes are seeded at a cell density of 0.5×10⁵. For each modifiedmRNA (mmRNA) to be transfected, modified mRNA: RNAIMAX™ is prepared asdescribed and mixed with the cells in the multi-well plate within aperiod of time, e.g., 6 hours, of cell seeding before cells had adheredto the tissue culture plate.

B. Forward Transfection

In a 24-well collagen-coated tissue culture plate, Keratinocytes areseeded at a cell density of 0.7×10⁵. For experiments performed in a96-well collagen-coated tissue culture plate, Keratinocytes are seededat a cell density of 0.3×10⁵. Keratinocytes are grown to a confluencyof >70% for over 24 hours. For each modified mRNA (mmRNA) to betransfected, modified mRNA: RNAIMAX™ is prepared as described andtransfected onto the cells in the multi-well plate over 24 hours aftercell seeding and adherence to the tissue culture plate.

C. Modified mRNA Translation Screen: G-CSF ELISA

Keratinocytes are grown in EPILIFE medium with Supplement S7 fromInvitrogen (Carlsbad, Calif.) at a confluence of >70%. One set ofkeratinocytes were reverse transfected with 300 ng of the chemicallymodified mRNA (mmRNA) complexed with RNAIMAX™ from Invitrogen. Anotherset of keratinocytes are forward transfected with 300 ng modified mRNAcomplexed with RNAIMAX™ from Invitrogen. The modified mRNA: RNAIMAX™complex is formed by first incubating the RNA with Supplement-freeEPILIFE® media in a 5× volumetric dilution for 10 minutes at roomtemperature.

In a second vial, RNAIMAX™ reagent was incubated with Supplement-freeEPILIFE® Media in 10× volumetric dilution for 10 minutes at roomtemperature. The RNA vial was then mixed with the RNAIMAX™ vial andincubated for 20-30 minutes at room temperature before being added tothe cells in a drop-wise fashion. Secreted human Granulocyte-ColonyStimulating Factor (G-CSF) concentration in the culture medium ismeasured at 18 hours post-transfection for each of the chemicallymodified mRNA in triplicate.

Secretion of Human G-CSF from transfected human keratinocytes isquantified using an ELISA kit from Invitrogen or R&D Systems(Minneapolis, Minn.) following the manufacturers recommendedinstructions.

D. Modified mRNA Dose and Duration: G-CSF ELISA

Keratinocytes are grown in EPILIFE® medium with Supplement S7 fromInvitrogen at a confluence of >70%. Keratinocytes are reversetransfected with either 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750ng, or 1500 ng modified mRNA complexed with the RNAIMAX™ from Invitrogen(Carlsbad, Calif.). The modified mRNA:RNAIMAX™ complex is formed asdescribed. Secreted human G-CSF concentration in the culture medium ismeasured at 0, 6, 12, 24, and 48 hours post-transfection for eachconcentration of each modified mRNA in triplicate. Secretion of humanG-CSF from transfected human keratinocytes is quantified using an ELISAkit from Invitrogen or R&D Systems following the manufacturersrecommended instructions.

Example 35 Split Dose Studies

Studies utilizing multiple subcutaneous or intramuscular injection sitesat one time point were designed and performed to investigate ways toincrease mmRNA drug exposure and improve protein production. In additionto detection of the expressed protein product, an assessment of thephysiological function of proteins was also determined through analyzingsamples from the animal tested.

Surprisingly, it has been determined that split dosing of mmRNA producesgreater protein production and phenotypic responses than those producedby single unit dosing or multi-dosing schemes.

The design of a single unit dose, multi-dose and split dose experimentinvolved using human erythropoietin (EPO) mmRNA (mRNA shown in SEQ IDNO: 33900; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1) administered in buffer alone. The dosing vehicle(F. buffer) consisted of 150 mM NaCl, 2 mM CaCl₂, 2 mM Na⁺ phosphate(1.4 mM monobasic sodium phosphate; 0.6 mM dibasic sodium phosphate),and 0.5 mM EDTA, pH 6.5. The pH was adjusted using sodium hydroxide andthe final solution was filter sterilized. The mmRNA was modified with5meC at each cytosine and pseudouridine replacement at each uridinesite.

Animals (n=5) were injected IM (intramuscular) for the single unit doseof 100 ug. For multi-dosing, two schedules were used, 3 doses of 100 ugand 6 doses of 100 ug. For the split dosing scheme, two schedules wereused, 3 doses at 33.3 ug and 6 doses of 16.5 ug mmRNA. Control dosinginvolved use of buffer only at 6 doses. Control mmRNA involved the useof luciferase mmRNA (IVT cDNA sequence shown in SEQ ID NO: 33906; mRNAsequence shown in SEQ ID NO: 33907, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) dosed 6 times at 100 ug. Blood and muscle tissue wereevaluated 13 hrs post injection.

Human EPO protein was measured in mouse serum 13 h post I.M. single,multi- or split dosing of the EPO mmRNA in buffer. Seven groups of mice(n=5 mice per group) were treated and evaluated. The results are shownin Table 59.

TABLE 59 Split dose study Avg. Poly- pmol/ peptide Dose mL per unitSplit- Dose of Total human drug ting Group Treatment mmRNA Dose EPO(pmol/ug) Factor 1 Human 1 × 100 ug  100 ug 14.3 .14 1 EPO mmRNA 2 Human3 × 100 ug  300 ug 82.5 .28 2 EPO mmRNA 3 Human 6 × 100 ug  600 ug 273.0.46 3.3 EPO mmRNA 4 Human 3 × 33.3 ug 100 ug 104.7 1.1 7.9 EPO mmRNA 5Human 6 × 16.5 ug 100 ug 127.9 1.3 9.3 EPO mmRNA 6 Luciferase 6 × 100ug  600 ug 0 — — mmRNA 7 Buffer — — 0 — — Alone

The splitting factor is defined as the product per unit drug divided bythe single dose product per unit drug (PUD). For example for treatmentgroup 2 the value 0.28 or product (EPO) per unit drug (mmRNA) is dividedby the single dose product per unit drug of 0.14. The result is 2.Likewise, for treatment group 4, the value 1.1 or product (EPO) per unitdrug (mmRNA) is divided by the single dose product per unit drug of0.14. The result is 7.9. Consequently, the dose splitting factor (DSF)may be used as an indicator of the efficacy of a split dose regimen. Forany single administration of a total daily dose, the DSF should be equalto 1. Therefore any DSF greater than this value in a split dose regimenis an indication of increased efficacy.

To determine the dose response trends, impact of injection site andimpact of injection timing, studies are performed. In these studies,varied doses of 1 ug, 5 ug, 10 ug, 25 ug, 50 ug, and values in betweenare used to determine dose response outcomes. Split dosing for a 100 ugtotal dose includes three or six doses of 1.6 ug, 4.2 ug, 8.3 ug, 16.6ug, or values and total doses equal to administration of the total doseselected.

Injection sites are chosen from the limbs or any body surface presentingenough area suitable for injection. This may also include a selection ofinjection depth to target the dermis (Intradermal), epidermis(Epidermal), subcutaneous tissue (SC) or muscle (IM). Injection anglewill vary based on targeted delivery site with injections targeting theintradermal site to be 10-15 degree angles from the plane of the surfaceof the skin, between 20-45 degrees from the plane of the surface of theskin for subcutaneous injections and angles of between 60-90 degrees forinjections substantially into the muscle.

Example 36 Quantification in Exosomes

The quantity and localization of the mmRNA of the present invention canbe determined by measuring the amounts (initial, timecourse, or residualbasis) in isolated exosomes. In this study, since the mmRNA aretypically codon-optimized and distinct in sequence from endogenous mRNA,the levels of mmRNA are quantitated as compared to endogenous levels ofnative or wild type mRNA by using the methods of Gibbings,PCT/IB2009/005878, the contents of which are incorporated herein byreference in their entirety.

In these studies, the method is performed by first isolating exosomes orvesicles preferably from a bodily fluid of a patient previously treatedwith a polynucleotide, primary construct or mmRNA of the invention, thenmeasuring, in said exosomes, the polynucleotide, primary construct ormmRNA levels by one of mRNA microarray, qRT-PCR, or other means formeasuring RNA in the art including by suitable antibody orimmunohistochemical methods.

Example 37 Effect of Modified mRNA on Cellular Viability, Cytotoxicityand Apoptosis

This experiment demonstrates cellular viability, cytotoxicity andapoptosis for distinct modified mRNA in-vitro transfected HumanKeratinocyte cells. Keratinocytes are grown in EPILIFE® medium withHuman Keratinocyte Growth Supplement in the absence of hydrocortisonefrom Invitrogen (Carlsbad, Calif.) at a confluence of >70%.Keratinocytes are reverse transfected with 0 ng, 46.875 ng, 93.75 ng,187.5 ng, 375 ng, 750 ng, 1500 ng, 3000 ng, or 6000 ng of modified mRNAcomplexed with RNAIMAX™ from Invitrogen. The modified mRNA:RNAIMAX™complex is formed. Secreted human G-CSF concentration in the culturemedium is measured at 0, 6, 12, 24, and 48 hours post-transfection foreach concentration of each modified m RNA in triplicate. Secretion ofhuman G-CSF from transfected human keratinocytes is quantified using anELISA kit from Invitrogen or R&D Systems following the manufacturersrecommended instructions.

Cellular viability, cytotoxicity and apoptosis is measured at 0, 12, 48,96, and 192 hours post-transfection using the APOTOX-GLO™ kit fromPromega (Madison, Wis.) according to manufacturer instructions.

Example 38 Detection of a Cellular Innate Immune Response to ModifiedmRNA Using an ELISA Assay

An enzyme-linked immunosorbent assay (ELISA) for Human Tumor NecrosisFactor-α (TNF-α), Human Interferon-β (IFN-β) and HumanGranulocyte-Colony Stimulating Factor (G-CSF) secreted from invitro-transfected Human Keratinocyte cells is tested for the detectionof a cellular innate immune response. Keratinocytes are grown inEPILIFE® medium with Human Keratinocyte Growth Supplement in the absenceof hydrocortisone from Invitrogen (Carlsbad, Calif.) at a confluenceof >70%. Secreted TNF-α keratinocytes are reverse transfected with 0 ng,93.75 ng, 1 87.5 ng, 375 ng, 750 ng, 1500 ng or 3000 ng of thechemically modified mRNA (mmRNA) complexed with RNAIMAX™ from Invitrogenas described in triplicate. Secreted TNF-α in the culture medium ismeasured 24 hours post-transfection for each of the chemically modifiedmRNA using an ELISA kit from Invitrogen according to the manufacturerprotocols.

Secreted IFN-β in the same culture medium is measured 24 hourspost-transfection for each of the chemically modified mRNA using anELISA kit from Invitrogen according to the manufacturer protocols.Secreted human G-CSF concentration in the same culture medium ismeasured at 24 hours post-transfection for each of the chemicallymodified mRNA. Secretion of human G-CSF from transfected humankeratinocytes is quantified using an ELISA kit from Invitrogen or R&DSystems (Minneapolis, Minn.) following the manufacturers recommendedinstructions. These data indicate which modified mRNA (mmRNA) arecapable eliciting a reduced cellular innate immune response incomparison to natural and other chemically modified polynucleotides orreference compounds by measuring exemplary type 1 cytokines TNF-α andIFN-β.

Example 39 Human Granulocyte-Colony Stimulating Factor (G-CSF) ModifiedmRNA-Induced Cell Proliferation Assay

Human keratinocytes are grown in EPILIFE® medium with Supplement S7 fromInvitrogen at a confluence of >70% in a 24-well collagen-coatedTRANSWELL® (Corning, Lowell, Mass.) co-culture tissue culture plate.Keratinocytes are reverse transfected with 750 ng of the indicatedchemically modified mRNA (mmRNA) complexed with RNAIMAX from Invitrogenas described in triplicate. The modified mRNA:RNAIMAX complex is formedas described. Keratinocyte media is exchanged 6-8 hourspost-transfection. 42-hours post-transfection, the 24-well TRANSWELL®plate insert with a 0.4 μm-pore semi-permeable polyester membrane isplaced into the human G-CSF modified mRNA-transfected keratinocytecontaining culture plate

Human myeloblast cells, Kasumi-1 cells or KG-1 (0.2×10⁵ cells), areseeded into the insert well and cell proliferation is quantified 42hours post-co-culture initiation using the CyQuant Direct CellProliferation Assay (Invitrogen, Carlsbad, Calif.) in a 100-120 μlvolume in a 96-well plate. Modified mRNA-encoding human G-CSF-inducedmyeloblast cell proliferation is expressed as a percent cellproliferation normalized to untransfected keratinocyte/myeloblastco-culture control wells. Secreted human G-CSF concentration in both thekeratinocyte and myeloblast insert co-culture wells is measured at 42hours post-co-culture initiation for each modified mRNA in duplicate.Secretion of human G-CSF is quantified using an ELISA kit fromInvitrogen following the manufacturer recommended instructions.

Transfected human G-CSF modified mRNA in human keratinocyte feeder cellsand untransfected human myeloblast cells are detected by RT-PCR. TotalRNA from sample cells is extracted and lysed using RNEASY® kit (Qiagen,Valencia, Calif.) according to the manufacturer instructions. Extractedtotal RNA is submitted to RT-PCR for specific amplification of modifiedmRNA-G-CSF using PROTOSCRIPT® M-MuLV Taq RT-PCR kit (New EnglandBioLabs, Ipswich, Mass.) according to the manufacturer instructions withhuman G-CSF-specific primers. RT-PCR products are visualized by 1.2%agarose gel electrophoresis.

Example 40 Co-Culture Assay

Modified mRNA comprised of chemically-distinct modified nucleotidesencoding human Granulocyte-Colony Stimulating Factor (G-CSF) maystimulate the cellular proliferation of a transfection incompetent cellin a co-culture environment. The co-culture includes a highlytransfectable cell type such as a human keratinocyte and a transfectionincompetent cell type such as a white blood cell (WBC). The modifiedmRNA encoding G-CSF are transfected into the highly transfectable cellallowing for the production and secretion of G-CSF protein into theextracellular environment where G-CSF acts in a paracrine-like manner tostimulate the white blood cell expressing the G-CSF receptor toproliferate. The expanded WBC population may be used to treatimmune-compromised patients or partially reconstitute the WBC populationof an immunosuppressed patient and thus reduce the risk of opportunisticinfections.

In another example, a highly transfectable cell such as a fibroblast aretransfected with certain growth factors support and simulate the growth,maintenance, or differentiation of poorly transfectable embryonic stemcells or induced pluripotent stem cells.

Example 41 Detection Assays of Human IgG Antibodies

A. ELISA Detection of Human IgG Antibodies

This example describes an ELISA for Human IgG from Chinese HamsterOvary's (CHO) and Human Embryonic Kidney (HEK, HER-2 Negative) 293 cellstransfected with human IgG modified mRNA (mmRNA). The Human EmbryonicEmbryonic Kidney (HEK) 293 are grown in CD 293 Medium with Supplement ofL-Glutamine from Invitrogen until they reach a confluence of 80-90%. TheCHO cells are grown in CD CHO Medium with Supplement of L-Glutamine,Hypoxanthine and Thymidine. In one aspect, 2×106 cells are transfectedwith 24 μg modified mRNA complexed with RNAIMAX™ from Invitrogen in a 75cm2 culture flask from Corning in 7 ml of medium. In another aspect,80,000 cells are transfected with 1 μg modified mRNA complexed withRNAIMAX™ from Invitrogen in a 24-well plate. The modified mRNA:RNAIMAX™complex is formed by incubating in a vial the mmRNA with either the CD293 or CD CHO medium in a 5× volumetric dilution for 10 minutes at roomtemperature. In a second vial, RNAIMAX™ reagent is incubated with CD 293medium or CD CHO medium in a 10× volumetric dilution for 10 minutes atroom temperature. The mmRNA vial is then mixed with the RNAIMAX™ vialand incubated for 20-30 minutes at room temperature before it is addedto the CHO or HEK cells in a drop-wise fashion. The culture supernatantsare stored at 4 degrees celsius. The concentration of the secreted humanIgG in the culture medium in the 24 μg mmRNA transfections is measuredat 12, 24, 36 hours post-transfection and the 1 μg mmRNA transfection ismeasured at 36 hours. Secretion of Trastuzumab from transfected HEK 293cells is quantified using an ELISA kit from Abcam (Cambridge, Mass.)following the manufacturers recommended instructions. The data showsthat a Humanized IgG antibody (such as Trastuzumab) mmRNA is capable ofbeing translated in HEK Cells and that Trastuzumab is secreted out ofthe cells and released into the extracellular environment. Furthermore,the data demonstrate that transfection of cells with mmRNA encodingTrastuzumab for the production of secreted protein can be scaled up to abioreactor or large cell culture conditions.

B. Western Detection of Modified mRNA Produced Human IgG Antibody

A Western Blot of CHO-K1 cells is co-transfected with 1 μg each of Heavyand Light Chain of Trastuzumab modified mRNA (mmRNA). CHO cells aregrown using standard protocols in 24-well plates. The cell supernatantsor cell lysates are collected 24 hours post-transfection, separated on a12% SDS-Page gel and transferred onto a nitrocellulose membrane usingthe IBOT® by Invitrogen (Carlsbad, Calif.). The cells are incubated witha first conjugation of a rabbit polyclonal antibody to Human IgGconjugated to DYLIGHT594 (ab96904, abcam, Cambridge, Mass.) and a secondconjugation of a goat polyclonal antibody to Rb IgG which is conjugatedto alkaline phosphatase. After incubation, the antibody is detectedusing Novex® alkaline phosphatase chromogenic substrate by Invitrogen(Carlsbad, Calif.).

C. Cell Immuno Staining of Modified mRNA Produced Trastuzumab andRituximab

CHO-K1 cells are co-transfected with 500 ng each of Heavy and LightChain of either Trastuzumab or Rituximab. Cells are grown in F-12KMedium from GIBCO® (Grand Island, N.Y.) and 10% FBS. Cells are fixedwith 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 inPBS for 5-10 minutes at room temperature and cells are washed 3 timeswith room temperature PBS. Trastuzumab and Rituximab staining isperformed using rabbit polyclonal antibody to Human IgG conjugated toDYLIGHT®594 (ab96904, abcam, Cambridge, Mass.) according to themanufacture's recommended dilutions. Nuclear DNA staining is performedwith DAPI dye from Invitrogen (Carlsbad, Calif.). The protein forTrastuzumab and Rituximab is translated and localized to the cytoplasmupon modified mRNA transfections. Pictures are taken 13 hourspost-transfection.

D. Binding Immunoblot Assay for Modified mRNA Produced Trastuzumab andRituximab

Trastuzumab and Rituximab are detected using a binding immunoblotdetection assay. Varying concentrations (100 ng/ul to 0 ng/ul) of theErB2 peptide (ab40048, abeam, Cambridge, Mass.), antigen for Trastuzumaband the CD20 peptide (ab97360, abeam, Cambridge, Mass.), antigen forRituximab are run on a 12% SDS-Page gel and transferred onto a membraneusing the iBlot from Invitrogen. The membranes are incubated for 1 hourwith their respective cell supernatants from CHO-K1 cells which areco-transfected with 500 ng each of Heavy and Light Chain of eitherTrastuzumab or Rituximab. The membranes are blocked with 1% BSA and asecondary anti-human IgG antibody conjugated to alkaline phosphatase(abcam, Cambridge, Mass.) is added. Antibody detection is conductedusing the NOVEX alkaline phosphatase chromogenic substrate by Invitrogen(Carlsbad, Calif.). The data shows that a humanized IgG antibodiesgenerated from modified mRNA is capable of recognizing and binding totheir respective antigens.

E. Cell Proliferation Assay

The SK-BR-3 cell line, an adherent cell line derived from a human breastadenocarcinoma, which overexpresses the HER2/neu receptor can be used tocompare the anti-proliferative properties of modified mRNA (mmRNA)generated Trastuzumab. Varying concentrations of purified Trastuzumabgenerated from modified mRNA and trastuzumab are be added to cellcultures, and their effects on cell growth are be assessed in triplicatecytotoxicity and viability assays.

Example 42 Bulk Transfection of Modified mRNA into Cell Culture

A. Cationic Lipid Delivery Vehicles

RNA transfections are carried out using RNAIMAX™ (Invitrogen, Carlsbad,Calif.) or TRANSIT-mRNA (Minis Bio, Madison, Wis.) cationic lipiddelivery vehicles. RNA and reagent are first diluted in Opti-MEM basalmedia (Invitrogen, Carlsbad, Calif.). 100 ng/uL RNA is diluted 5× and 5μL of RNAIMax per μg of RNA is diluted 10×. The diluted components arepooled and incubated 15 minutes at room temperature before they aredispensed to culture media. For TRANSIT-mRNA transfections, 100 ng/uLRNA is diluted 10× in Opti-MEM and BOOST reagent is added (at aconcentration of 2 μL per μg of RNA), TRANSIT-mRNA is added (at aconcentration of 2 μL per μg of RNA), and then the RNA-lipid complexesare delivered to the culture media after a 2-minute incubation at roomtemperature. RNA transfections are performed in Nutristem xenofree hESmedia (Stemgent, Cambridge, Mass.) for RiPS derivations, Dermal CellBasal Medium plus Keratinocyte Growth Kit (ATCC) for keratinocyteexperiments, and Opti-MEM plus 2% FBS for all other experiments.Successful introduction of a modified mRNA (mmRNA) into host cells canbe monitored using various known methods, such as a fluorescent marker,such as Green Fluorescent Protein (GFP). Successful transfection of amodified mRNA can also be determined by measuring the protein expressionlevel of the target polypeptide by e.g., Western Blotting orimmunocytochemistry. Similar methods may be followed for large volumescale-up to multi-liter (5-10,000 L) culture format following similarRNA-lipid complex ratios.

B. Electroporation Delivery of Exogenous Synthetic mRNA Transcripts

Electroporation parameters are optimized by transfecting MRC-5fibroblasts with in vitro synthetic modified mRNA (mmRNA) transcriptsand measuring transfection efficiency by quantitative RT-PCR withprimers designed to specifically detect the exogenous transcripts.Discharging a 150 uF capacitor charged to F into 2.5×10⁶ cells suspendedin 50 μl of Opti-MEM (Invitrogen, Carlsbad, Calif.) in a standardelectroporation cuvette with a 2 mm gap is sufficient for repeateddelivery in excess of 10,000 copies of modified mRNA transcripts percell, as determined using the standard curve method, while maintaininghigh viability (>70%). Further experiments may reveal that the voltagerequired to efficiently transfect cells with mmRNA transcripts candepend on the cell density during electroporation. Cell density may varyfrom 1×10⁶ cell/50 μl to a density of 2.5×10⁶ cells/50 μl and requirefrom 110V to 145V to transfect cells with similar efficiencies measuredin transcript copies per cell. Large multi-liter (5-10,000 L)electroporation may be performed similar to large volume flowelectroporation strategies similar to methods described with the abovedescribed constraints (Li et al., 2002; Geng et al., 2010).

Example 43 In Vivo Delivery Using Lipoplexes

A. Human EPO Modified RNA Lipoplex

A formulation containing 100 μg of modified human erythropoietin mRNA(mRNA shown in SEQ ID NO: 33897; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) (EPO; fully modified5-methylcytosine; N1-methylpseudouridine) was lipoplexed with 30% byvolume of RNAIMAX™ (Lipoplex-h-Epo-46; Generation 2 or Gen2) in 50-70 uLdelivered intramuscularly to four C57/BL6 mice. Other groups consistedof mice receiving an injection of the lipoplexed modified luciferasemRNA (Lipoplex-luc) (IVT cDNA sequence shown in SEQ ID NO: 33906; mRNAsequence shown in SEQ ID NO: 33907, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) which served as a control containing 100 μg of modifiedluciferase mRNA was lipoplexed with 30% by volume of RNAiMAX™ or micereceiving an injection of the formulation buffer as negative control ata dose volume of 65 ul. 13 hours after the intramuscular injection,serum was collected from each mouse to measure the amount of human EPOprotein in the mouse serum by human EPO ELISA and the results are shownin Table 60.

TABLE 60 Human EPO Production (IM Injection Route) Formualtion AverageLipoplex-h-Epo-46 251.95 Lipoplex-Luc 0 Formulation Buffer 0

B. Human G-CSF Modified RNA Lipoplex

A formulation containing 100 μg of one of the two types of modifiedhuman G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 33897; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) (G-CSFfully modified with 5-methylcytosine and pseudouridine (G-CSF) or G-CSFfully modified with 5-methylcytosine and N1-methyl-pseudouridine(G-CSF-N1) lipoplexed with 30% by volume of RNAIMAX™ and delivered in150 uL intramuscularly (I.M), in 150 uL subcutaneously (S.C) and in 225uL intravenously (I.V) to C57/BL6 mice. Three control groups wereadministered either 100 μg of modified luciferase mRNA (IVT cDNAsequence shown in SEQ ID NO: 33906; mRNA sequence shown in SEQ ID NO:33907, polyA tail of approximately 160 nucleotides not shown insequence, 5′ cap, Cap1, fully modified with 5-methylcytosine at eachcytosine and pseudouridine replacement at each uridine site)intramuscularly (Luc-unsp I.M.) or 150 μg of modified luciferase mRNAintravenously (Luc-unsp I.V.) or 150 uL of the formulation bufferintramuscularly (Buffer I.M.). 6 hours after administration of aformulation, serum was collected from each mouse to measure the amountof human G-CSF protein in the mouse serum by human G-CSF ELISA and theresults are shown in Table 61.

These results demonstrate that both 5-methylcytosine/pseudouridine and5-methylcytosine/N1-methylpseudouridine modified human G-CSF mRNA canresult in specific human G-CSF protein expression in serum whendelivered via I.V. or I.M. route of administration in a lipoplexformulation.

TABLE 61 Human G-CSF in Serum (I.M., I.V., S.C. Injection Route)Formulation Route G-CSF (pg/ml) G-CSF I.M. 85.6 G-CSF N1 I.M. 40.1 G-CSFS.C. 3.9 G-CSF N1 S.C. 0.0 G-CSF I.V. 31.0 G-CSF N1 I.V. 6.1 Luc-unspI.M. 0.0 Luc-unsp I.V. 0.0 Buffer I.M. 0.0

C. Human G-CSF Modified RNA Lipoplex Comparison

A formulation containing 100 μg of either modified human G-CSF mRNAlipoplexed with 30% by volume of RNAIMAX™ with a 5-methylcytosine (5mc)and a pseudouridine (ψ) modification (G-CSF-Gen1-Lipoplex), modifiedhuman G-CSF mRNA with a 5mc and ψ modification in saline(G-CSF-Gen1-Saline), modified human G-CSF mRNA with aN1-5-methylcytosine (N1-5mc) and a ψ modification lipoplexed with 30% byvolume of RNAIMAX™ (G-CSF-Gen2-Lipoplex), modified human G-CSF mRNA witha N1-5mc and ψ modification in saline (G-CSF-Gen2-Saline), modifiedluciferase with a 5mc and ψ modification lipoplexed with 30% by volumeof RNAIMAX™ (Luc-Lipoplex), or modified luciferase mRNA with a 5mc and ψmodification in saline (Luc-Saline) was delivered intramuscularly (I.M.)or subcutaneously (S.C.) and a control group for each method ofadministration was giving a dose of 80 uL of the formulation buffer (F.Buffer) to C57/BL6 mice. 13 hours post injection serum and tissue fromthe site of injection were collected from each mouse and analyzed byG-CSF ELISA to compare human G-CSF protein levels. The results of thehuman G-CSF protein in mouse serum from the intramuscularadministration, and the subcutaneous administration results are shown inTable 62.

These results demonstrate that 5-methylcytosine/pseudouridine and5-methylcytosine/N1-methylpseudouridine modified human G-CSF mRNA canresult in specific human G-CSF protein expression in serum whendelivered via I.M. or S.C. route of administration whether in a salineformulation or in a lipoplex formulation. As shown in Table 62,5-methylcytosine/N1-methyl-pseudouridine modified human G-CSF mRNAgenerally demonstrates increased human G-CSF protein production relativeto 5-methylcytosine/pseudouridine modified human G-CSF mRNA.

TABLE 62 Human G-CSF Protein in Mouse Serum G-CSF (pg/ml) FormulationI.M. Injection Route S.C. Injenction Route G-CSF-Gen1-Lipoplex 13.98842.855 G-CSF-Gen1-saline 9.375 4.614 G-CSF-Gen2-lipoplex 75.572 32.107G-CSF-Gen2-saline 20.190 45.024 Luc lipoplex 0 3.754 Luc saline 0.0748 0F. Buffer 4.977 2.156

D. mCherry Modified RNA Lipoplex Comparison

Intramuscular and Subcutaneous Administration

A formulation containing 100 μg of either modified mCherry mRNA (mRNAsequence shown in SEQ ID NO: 33898; polyA tail of approximately 160nucleotides not shown in sequence; 5′cap, Cap1) lipoplexed with 30% byvolume of RNAIMAX™ or modified mCherry mRNA in saline is deliveredintramuscularly and subcutaneously to mice. A formulation buffer is alsoadministered to a control group of mice either intramuscularly orsubcutaneously. The site of injection on the mice may be collected 17hours post injection for sectioning to determine the cell type(s)responsible for producing protein.

Intravitreal Administration

A formulation containing 10 μg of either modified mCherry mRNAlipoplexed with RNAIMAX™, modified mCherry mRNA in a formulation buffer,modified luciferase mRNA lipoplexed with RNAMAX™, modified luciferasemRNA in a formulation buffer can be administered by intravitrealinjection (IVT) in rats in a dose volume of 5 μl/eye. A formulationbuffer is also administered by IVT to a control group of rats in a dosevolume of 5 μl/eye. Eyes from treated rats can be collected after 18hours post injection for sectioning and lysating to determine whethermmRNA can be effectively delivered in vivo to the eye and result inprotein production, and to also determine the cell type(s) responsiblefor producing protein in vivo.

Intranasal Administration

A formulation containing 100 μg of either modified mCherry mRNAlipoplexed with 30% by volume of RNAIMAX™, modified mCherry mRNA insaline, modified luciferase mRNA lipoplexed with 30% by volume ofRNAIMAX™ or modified luciferase mRNA in saline is deliveredintranasally. A formulation buffer is also administered to a controlgroup intranasally. Lungs may be collected about 13 hours postinstillation for sectioning (for those receiving mCherry mRNA) orhomogenization (for those receiving luciferase mRNA). These samples willbe used to determine whether mmRNA can be effectively delivered in vivoto the lungs and result in protein production, and to also determine thecell type(s) responsible for producing protein in vivo.

Example 44 In Vivo Delivery Using Varying Lipid Ratios

Modified mRNA was delivered to C57/BL6 mice to evaluate varying lipidratios and the resulting protein expression. Formulations of 100 μgmodified human EPO mRNA (mRNA shown in SEQ ID NO: 33900; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) lipoplexed with 10%,30% or 50% RNAIMAX™, 100 μg modified luciferase mRNA (IVT cDNA sequenceshown in SEQ ID NO: 33906; mRNA sequence shown in SEQ ID NO: 33907,polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site) lipoplexed with 10%, 30%or 50% RNAIMAX™ or a formulation buffer were administeredintramuscularly to mice in a single 70 μl dose. Serum was collected 13hours post injection to undergo a human EPO ELISA to determine the humanEPO protein level in each mouse. The results of the human EPO ELISA,shown in Table 63, show that modified human EPO expressed in the muscleis secreted into the serum for each of the different percentage ofRNAIMAX™.

TABLE 63 Human EPO Protein in Mouse Serum (IM Injection Route)Formulation EPO (pg/ml) Epo + 10% RNAiMAX 11.4 Luc + 10% RNAiMAX 0 Epo +30% RNAiMAX 27.1 Luc + 30% RNAiMAX 0 Epo + 50% RNAiMAX 19.7 Luc + 50%RNAiMAX 0 F. Buffer 0

Example 45 Intramuscular and Subcutaneous In Vivo Delivery in Mammals

Modified human EPO mRNA (mRNA sequence shown in SEQ ID NO: 33900; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) formulatedin saline was delivered to either C57/BL6 mice or Sprague-Dawley rats toevaluate the dose dependency on human EPO production. Rats wereintramuscularly injected with 50 μl of the modified human EPO mRNA(h-EPO), modified luciferase mRNA (Luc) (IVT cDNA sequence shown in SEQID NO: 33906; mRNA sequence shown in SEQ ID NO: 33907, polyA tail ofapproximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fullymodified with 5-methylcytosine at each cytosine and pseudouridinereplacement at each uridine site) or the formulation buffer (F.Buffer)as described in the dosing chart Table 64.

Mice were intramuscularly or subcutaneously injected with 50 μl of themodified human EPO mRNA (h-EPO), modified luciferase mRNA (Luc) or theformulation buffer (F.Buffer) as described in the dosing chart Table 65.13 hours post injection blood was collected and serum was analyzed todetermine the amount human EPO for each mouse or rat. The average andgeometric mean in pg/ml for the rat study are also shown in Table 64.

TABLE 64 Rat Study Geometric- Dose Avg. mean Group (ug) R#1 R#2 R#3 R#4R#5 R#6 pg/ml pg/ml h-EPO G#1 150 61.8 86.3 69.9 55.2 59 74.2 67.7 67.1h-EPO G#2 100 69.4 77.8 48.2 17.6 101.9 161.5 79.4 66.9 h-EPO G#3 50143.6 60.9 173.4 145.9 61.5 23.9 101.5 85.4 h-EPO G#4 10 7.8 11.8 30.936.2 40.6 150.3 46.3 31.2 h-EPO G#5 1 9.1 35.8 — 46.2 18.1 34.1 28.725.4 Luc G#6 100 34.1 36.5 13.5 13.7 — — 24.5 22.4 F. Buffer G#7 — 14.718.5 21.2 20.3 — — 18.7 18.5

TABLE 65 Mouse Study Average Level in serum Route Treatment Group Dosepg/ml IM h-EPO 1 100 μg 96.2 IM h-EPO 2  50 μg 63.5 IM h-EPO 3  25 μg18.7 IM h-EPO 4  10 μg 25.9 IM h-EPO 5  1 μg 2.6 IM Luc 6 100 μg 0 IM F.Buffer 7 — 1.0 SC h-EPO 1 100 μg 72.0 SC Luc 2 100 μg 26.7 SC F. Buffer3 — 17.4

Example 46 Duration of Activity after Intramuscular In Vivo Delivery inRats

Modified human EPO mRNA (mRNA sequence shown in SEQ ID NO: 33900; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) formulatedin formulation buffer was delivered to Sprague-Dawley rats to determinethe duration of the dose response. Rats were intramuscularly injectedwith 50 μl of the modified human EPO mRNA (h-EPO), modified luciferasemRNA (IVT cDNA sequence shown in SEQ ID NO: 33906; mRNA sequence shownin SEQ ID NO: 33907, polyA tail of approximately 160 nucleotides notshown in sequence, 5′ cap, Cap1, fully modified with 5-methylcytosine ateach cytosine and pseudouridine replacement at each uridine site) (Luc)or the formulation buffer (F.Buffer) as described in the dosing chartTable 66. The rats were bled 2, 6, 12, 24, 48 and 72 hours after theintramuscular injection to determine the concentration of human EPO inserum at a given time. The average and geometric mean in pg/ml for thisstudy are also shown in Table 66.

TABLE 66 Dosing Chart Dose Avg. Geometric-mean Group (ug) pg/ml (pg/ml)h-EPO 2 hour 100 59.6 58.2 h-EPO 6 hour 100 68.6 55.8 h-EPO 12 hour 10087.4 84.5 h-EPO 24 hour 100 108.6 95.3 h-EPO 48 hour 100 77.9 77.0 h-EPO72 hour 100 80.1 75.8 Luc 24, 48 100 37.2 29.2 and 72 hour F.Buffer 24,48 — 48.9 10.4 and 72 hour

Example 47 Routes of Administration

Further studies were performed to investigate dosing using differentroutes of administration. Following the protocol outlined in Example 35,4 mice per group were dosed intramuscularly (I.M.), intravenously (IV)or subcutaneously (S.C.) by the dosing chart outlined in Table 67. Serumwas collected 13 hours post injection from all mice, tissue wascollected from the site of injection from the intramuscular andsubcutaneous group and the spleen, liver and kidneys were collected fromthe intravenous group. The results from the intramuscular group and thesubcutaneous group results are shown in Table 68.

TABLE 67 Dosing Chart Dose of Total Dosing Group Treatment Route mmRNADose Vehicle  1 Lipoplex-human EPO I.M. 4 × 100 ug + 4 × 70 ul LipoplexmmRNA 30% Lipoplex  2 Lipoplex-human EPO I.M. 4 × 100 ug 4 × 70 ulBuffer mmRNA  3 Lipoplex-human EPO S.C. 4 × 100 ug + 4 ×70 ul LipoplexmmRNA 30% Lipoplex  4 Lipoplex-human EPO S.C. 4 × 100 ug 4 × 70 ulBuffer mmRNA  5 Lipoplex-human EPO I.V. 200 ug + 30% 140 ul LipoplexmmRNA Lipoplex  6 Lipoplexed-Luciferase I.M. 100 ug + 30% 4 × 70 ulLipoplex mmRNA Lipoplex  7 Lipoplexed-Luciferase I.M. 100 ug 4 × 70 ulBuffer mmRNA  8 Lipoplexed-Luciferase S.C. 100 ug + 30% 4 × 70 ulLipoplex mmRNA Lipoplex  9 Lipoplexed-Luciferase S.C. 100 ug 4 × 70 ulBuffer mmRNA 10 Lipoplexed-human EPO I.V. 200 ug + 30% 140 ul LipoplexmmRNA Lipoplex 11 Formulation Buffer I.M. 4 × multi 4 ×70 ul Bufferdosing

TABLE 68 Human EPO Protein in Mouse Serum (I.M. Injection Route) EPO PO(pg/ml) Formulation I.M. Injection Route S.C. Injection RouteEpo-Lipoplex  67.115  2.154 Luc-Lipoplex 0 0 Epo-Saline 100.891 11.37Luc-Saline 0 0 Formulation Buffer 0 0

Example 48 Rapidly Eliminated Lipid Nanoparticle (reLNP) Studies

A. Formulation of Modified RNA reLNPs

Solutions of synthesized lipid, 1,2-distearoyl-3-phosphatidylcholine(DSPC) (Avanti Polar Lipids, Alabaster, Ala.), cholesterol(Sigma-Aldrich, Taufkirchen, Germany), andα-[3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-ω-methoxy-polyoxyethylene(PEG-c-DOMG) (NOF, Bouwelven, Belgium) are prepared and stored at −20°C. The synthesized lipid is selected from DLin-DMA with an internalester, DLin-DMA with a terminal ester, DLin-MC3-DMA-internal ester, andDLin-MC3-DMA with a terminal ester. The reLNPs are combined to yield amolar ratio of 50:10:38.5:1.5 (reLNP:DSPC:Cholesterol:PEG-c-DOMG).Formulations of the reLNPs and modified mRNA are prepared by combiningthe lipid solution with the modified mRNA solution at total lipid tomodified mRNA weight ratio of 10:1, 15:1, 20:1 and 30:1.

B. Characterization of Formulations

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) is used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the modified mRNA nanoparticles in 1×PBSin determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy is used to determine the concentrationof modified mRNA nanoparticle formulation. After mixing, the absorbancespectrum of the solution is recorded between 230 nm and 330 nm on a DU800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea,Calif.). The modified RNA concentration in the nanoparticle formulationis calculated based on the extinction coefficient of the modified RNAused in the formulation and on the difference between the absorbance ata wavelength of 260 nm and the baseline value at a wavelength of 330 nm.

QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.)is used to evaluate the encapsulation of modified RNA by thenanoparticle. The samples are diluted, transferred to a polystyrene 96well plate, then either a TE buffer or a 2% Triton X-100 solution isadded. The plate is incubated and the RIBOGREEN® reagent is diluted inTE buffer, and of this solution is added to each well. The fluorescenceintensity is measured using a fluorescence plate reader (Wallac Victor1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) The fluorescencevalues of the reagent blank are subtracted from each of the samples andthe percentage of free modified RNA is determined by dividing thefluorescence intensity of the intact sample by the fluorescence value ofthe disrupted sample.

C. In Vitro Incubation

Human embryonic kidney epithelial (HEK293) and hepatocellular carcinomaepithelial (HepG2) cells (LGC standards GmbH, Wesel, Germany) are seededon 96-well plates (Greiner Bio-one GmbH, Frickenhausen, Germany) andplates for HEK293 cells are precoated with collagen type1. HEK293 areseeded at a density of about 30,000 and HepG2 are seeded at a density ofabout 35,000 cells per well in 100 μl cell culture medium. Formulationscontaining mCherry mRNA (mRNA sequence shown in SEQ ID NO: 33898; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1) are added directly after seeding the cells and incubated. ThemCherry cDNA with the T7 promoter, 5′untranslated region (UTR) and 3′UTR used in in vitro transcription (IVT) is given in SEQ ID NO: 33899.

Cells are harvested by transferring the culture media supernatants to a96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg,Germany). Cells are trypsinized with ½ volume Trypsin/EDTA (Biochrom AG,Berlin, Germany), pooled with respective supernatants and fixed byadding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5%formaldehyde (Merck, Darmstadt, Germany). Samples are then submitted toa flow cytometer measurement with an excitation laser and a filter forPE-Texas Red in a LSRII cytometer (Beckton Dickinson GmbH, Heidelberg,Germany). The mean fluorescence intensity (MFI) of all events and thestandard deviation of four independent wells are presented in forsamples analyzed.

D. In Vivo Formulation Studies

Mice are administered intravenously a single dose of a formulationcontaining a modified mRNA and a reLNP. The modified mRNA administeredto the mice is selected from G-CSF (mRNA sequence shown in SEQ ID NO:33897; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1), Factor IX (mRNA shown in SEQ ID NO: 33901; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1) or mCherry (mRNA sequence shown in SEQ ID NO: 33898; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1).

The mice are injected with 100 ug, 10 ug or 1 ug of the formulatedmodified mRNA and are sacrificed 8 hours after they are administered theformulation. Serum from the mice administered formulations containinghuman G-CSF modified mRNA are measured by specific G-CSF ELISA and serumfrom mice administered human Factor IX modified RNA is analyzed byspecific factor IX ELISA or chromogenic assay. The liver and spleen fromthe mice administered with mCherry modified mRNA are analyzed byimmunohistochemistry (IHC) or fluorescence-activated cell sorting(FACS). As a control, a group of mice are not injected with anyformulation and their serum and tissue are collected analyzed by ELISA,FACS and/or IHC.

Example 49 In Vitro Transfection of VEGF-A

Human vascular endothelial growth factor-isoform A (VEGF-A) modifiedmRNA (mRNA sequence shown in SEQ ID NO: 33910; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) wastransfected via reverse transfection in Human Keratinocyte cells in 24multi-well plates. Human Keratinocytes cells were grown in EPILIFE®medium with Supplement S7 from Invitrogen (Carlsbad, Calif.) until theyreached a confluence of 50-70%. The cells were transfected with 0,46.875, 93.75, 187.5, 375, 750, and 1500 ng of modified mRNA (mmRNA)encoding VEGF-A which had been complexed with RNAIMAX™ from Invitrogen(Carlsbad, Calif.). The RNA:RNAIMAX™ complex was formed by firstincubating the RNA with Supplement-free EPILIFE® media in a 5×volumetric dilution for 10 minutes at room temperature. In a secondvial, RNAIMAX™ reagent was incubated with Supplement-free EPILIFE® Mediain a 10× volumetric dilution for 10 minutes at room temperature. The RNAvial was then mixed with the RNAIMAX™ vial and incubated for 20-30minutes at room temperature before being added to the cells in adrop-wise fashion.

The fully optimized mRNA encoding VEGF-A (mRNA sequence shown in SEQ IDNO: 33910; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1) transfected with the Human Keratinocyte cellsincluded modifications during translation such as natural nucleosidetriphosphates (NTP), pseudouridine at each uridine site and5-methylcytosine at each cytosine site (pseudo-U/5mC), andN1-methyl-pseudouridine at each uridine site and 5-methylcytosine ateach cytosine site (N1-methyl-Pseudo-U/5mC). Cells were transfected withthe mmRNA encoding VEGF-A and secreted VEGF-A concentration (ρg/ml) inthe culture medium was measured at 6, 12, 24, and 48 hourspost-transfection for each of the concentrations using an ELISA kit fromInvitrogen (Carlsbad, Calif.) following the manufacturers recommendedinstructions. These data, shown in Table 69, show that modified mRNAencoding VEGF-A is capable of being translated in Human Keratinocytecells and that VEGF-A is transported out of the cells and released intothe extracellular environment.

TABLE 69 VEGF-A Dosing and Protein Secretion Dose 6 hours 12 hours 24hours 48 hours (ng) (pg/ml) (pg/ml) (pg/ml) (pg/ml) VEGF-A DoseContaining Natural NTPs 46.875 10.37 18.07 33.90 67.02 93.75 9.79 20.5441.95 65.75 187.5 14.07 24.56 45.25 64.39 375 19.16 37.53 53.61 88.28750 21.51 38.90 51.44 61.79 1500 36.11 61.90 76.70 86.54 VEGF-A DoseContaining Pseudo-U/5mC 46.875 10.13 16.67 33.99 72.88 93.75 11.00 20.0046.47 145.61 187.5 16.04 34.07 83.00 120.77 375 69.15 188.10 448.50392.44 750 133.95 304.30 524.02 526.58 1500 198.96 345.65 426.97 505.41VEGF-A Dose Containing N1-methyl-Pseudo-U/5mC 46.875 0.03 6.02 27.65100.42 93.75 12.37 46.38 121.23 167.56 187.5 104.55 365.71 1025.411056.91 375 605.89 1201.23 1653.63 1889.23 750 445.41 1036.45 1522.861954.81 1500 261.61 714.68 1053.12 1513.39

Example 50 In Vivo Studies of Factor IX

Human Factor IX mmRNA (Gen1; fully modified 5-methylcytosine andpseudouridine) formulated in formulation buffer was delivered to micevia intramuscular injection. The results demonstrate that Factor IXprotein was elevated in serum as measured 13 hours after administration.

In this study, mice (N=5 for Factor IX, N=3 for Luciferase or Buffercontrols) were intramuscularly injected with 50 μl of the Factor IXmmRNA (mRNA sequence shown in SEQ ID NO: 33901; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1),Luciferase (IVT cDNA sequence shown in SEQ ID NO: 33906; mRNA sequenceshown in SEQ ID NO: 33907, polyA tail of approximately 160 nucleotidesnot shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) or the formulation buffer (F.Buffer) at 2×100 ug/mouse.The mice were bled at 13 hours after the intramuscular injection todetermine the concentration of human the polypeptide in serum in pg/mL.The results revealed that administration of Factor IX mmRNA resulted inlevels of 1600 pg/mL at 13 hours as compared to less than 100 pg/mL ofFactor IX for either Luciferase or buffer control administration.

Example 51 Multi-Site Administration: Intramuscular and Subcutaneous

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 33897;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1) modified as either Gen1 or Gen2 (5-methylcytosine (5mc) anda pseudouridine (ψ) modification, G-CSF-Gen1; or N1-5-methylcytosine(N1-5mc) and a ψ modification, G-CSF-Gen2) and formulated in formulationbuffer were delivered to mice via intramuscular (IM) or subcutaneous(SC) injection. Injection of four doses or 2×50 ug (two sites) daily forthree days (24 hrs interval) was performed. The fourth dose wasadministered 6 hrs before blood collection and CBC analysis. Controlsincluded Luciferase (IVT cDNA sequence shown in SEQ ID NO: 33906; mRNAsequence shown in SEQ ID NO: 33907, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) or the formulation buffer (F.Buffer). The mice were bledat 72 hours after the first mRNA injection (6 hours after the lastmodified mRNA dose) to determine the effect of mRNA-encoded human G-CSFon the neutrophil count. The dosing regimen is shown in Table 70 as arethe resulting neutrophil counts (thousands/uL). In Table 70, an asterisk(*) indicates statistical significance at p<0.05. For intramuscularadministration, the data reveal a four fold increase in neutrophil countabove control at day 3 for the Gen1 G-CSF mRNA and a two fold increasefor the Gen2 G-CSF mmRNA. For subcutaneous administration, the datareveal a two fold increase in neutrophil count above control at day 3for the Gen2 G-CSF mRNA.

These data demonstrate that both 5-methylcytidine/pseudouridine and5-methylcytidine/N1-methylpseudouridine-modified mRNA can bebiologically active, as evidenced by specific increases in bloodneutrophil counts.

TABLE 70 Dosing Regimen Dose Dose Vol. Neutro- (μg/ (μl/ Dosing phil Gr.Treatment Route N = mouse) mouse) Vehicle Thous/uL 1 G-CSF I.M 5 2 × 50ug 50 F. buffer  840* (Gen1) (four doses) 2 G-CSF S.C 5 2 × 50 ug 50 F.buffer 430 (Gen1) (four doses) 3 G-CSF I.M 5 2 × 50 ug 50 F. buffer 746* (Gen2) (four doses) 4 G-CSF S.C 5 2 × 50 ug 50 F. buffer 683(Gen2) (four doses) 5 Luc I.M. 5 2 × 50 ug 50 F. buffer 201 (Gen1) (fourdoses) 6 Luc S.C. 5 2 × 50 ug 50 F. buffer 307 (Gen1) (four doses) 7 LucI.M 5 2 × 50 ug 50 F. buffer 336 (Gen2) (four doses) 8 Luc S.C 5 2 × 50ug 50 F. buffer 357 (Gen2) (four doses) 9 F. Buffer I.M 4 0 50 F. buffer245 (four doses) 10 F. Buffer S.C. 4 0 50 F. buffer 509 (four doses) 11Untreated — 4 — 312

Example 52 Intravenous Administration

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 33897;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1) modified with 5-methylcytosine (5mc) and a pseudouridine(ψ) modification (Gen1); or having no modifications and formulated in10% lipoplex (RNAiMax) were delivered to mice at a dose of 50 ug RNA andin a volume of 100 ul via intravenous (IV) injection at days 0, 2 and 4.Neutrophils were measured at days 1, 5 and 8. Controls includednon-specific mammalian RNA or the formulation buffer alone (F.Buffer).The mice were bled at days 1, 5 and 8 to determine the effect ofmodified mRNA-encoded human G-CSF to increase neutrophil count. Thedosing regimen is shown in Table 71 as are the resulting neutrophilcounts (thousands/uL; K/uL).

For intravenous administration, the data reveal a four to five foldincrease in neutrophil count above control at day 5 with G-CSF modifiedmRNA but not with unmodified G-CSF mRNA or non-specific controls. Bloodcount returned to baseline four days after the final injection. No otherchanges in leukocyte populations were observed. In Table 71, an asterisk(*) indicates statistical significance at p<0.001 compared to buffer.

These data demonstrate that lipoplex-formulated5-methylcytidine/pseudouridine-modified mRNA can be biologically active,when delivered through an I.V. route of administration as evidenced byspecific increases in blood neutrophil counts. No other cell subsetswere significantly altered. Unmodified G-CSF mRNA similarly administeredshowed no pharmacologic effect on neutrophil counts.

TABLE 71 Dosing Regimen Dose Neutro- Vol. Dosing phil Gr. Day TreatmentN = (μl/mouse) Vehicle K/uL 1 1 G-CSF (Gen1) 5 100 10% lipoplex 2.91 2 5G-CSF (Gen1) 5 100 10% lipoplex  5.32* 3 8 G-CSF (Gen1) 5 100 10%lipoplex 2.06 4 1 G-CSF 5 100 10% lipoplex 1.88 (no modification) 5 5G-CSF 5 100 10% lipoplex 1.95 (no modification) 6 8 G-CSF 5 100 10%lipoplex 2.09 (no modification) 7 1 RNA control 5 100 10% lipoplex 2.908 5 RNA control 5 100 10% lipoplex 1.68 9 8 RNA control 4 100 10%lipoplex 1.72 10 1 F. Buffer 4 100 10% lipoplex 2.51 11 5 F. Buffer 4100 10% lipoplex 1.31 12 8 F. Buffer 4 100 10% lipoplex 1.92

Example 53 Saline Formulation: Intramuscular Administration

A. Protein Production

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 33897;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1) and human EPO mmRNA (mRNA sequence shown in SEQ ID NO:33900; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1); G-CSF modified mRNA (modified with5-methylcytosine (5mc) and pseudouridine (ψ)) and EPO modified mRNA(modified with N1-5-methylcytosine (N1-5mc) and ψ modification), wereformulated in formulation buffer (150 mM sodium chloride, 2 mM calciumchloride, 2 mM phosphate, 0.5 mM EDTA at a pH of 6.5) and delivered tomice via intramuscular (IM) injection at a dose of 100 ug.

Controls included Luciferase (IVT cDNA sequence shown in SEQ ID NO:33906; mRNA sequence shown in SEQ ID NO: 33907, polyA tail ofapproximately 160 nucleotides not shown in sequence, 5′ cap, Cap1, fullymodified with 5-methylcytosine at each cytosine and pseudouridinereplacement at each uridine site) or the formulation buffer (F.Buffer).The mice were bled at 13 hours after the injection to determine theconcentration of the human polypeptide in serum in pg/mL. (G-CSF groupsmeasured human G-CSF in mouse serum and EPO groups measured human EPO inmouse serum). The data are shown in Table 72.

TABLE 72 Dosing Regimen Average Protein Dose Product Vol. Dosing pg/mL,Group Treatment N = (μl/mouse) Vehicle serum G-CSF G-CSF 5 50 Saline19.8 G-CSF Luciferase 5 50 Saline 0.5 G-CSF F. buffer 5 50 F. buffer 0.5EPO EPO 5 50 Saline 191.5 EPO Luciferase 5 50 Saline 15.0 EPO F. bufferF. buffer 4.8

B. Dose Response

Human EPO modified mRNA (mRNA sequence shown in SEQ ID NO: 33900; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) wereformulated in formulation buffer and delivered to mice via intramuscular(IM) injection.

Controls included Luciferase (mRNA sequence shown in SEQ ID NO: 33906,polyA tail of approximately 160 nucleotides not shown in sequence,5′cap, Cap1, fully modified with 5-methylcytosine and pseudouridine) orthe formulation buffer (F.Buffer). The mice were bled at 13 hours afterthe injection to determine the concentration of the human polypeptide inserum in pg/mL. The dose and expression are shown in Table 73.

TABLE 73 Dosing Regimen and Expression Average Protein Dose Product Vol.pg/mL, Treatment (μl/mouse) serum EPO 100 96.2 EPO  50 63.5 EPO  25 18.7EPO  10 25.9 EPO   1 2.6 Luciferase 100 0.0 F. buffer 100 1.0

Example 54 EPO Multi-Dose/Multi-Administration

Studies utilizing multiple intramuscular injection sites at one timepoint were designed and performed.

The design of a single multi-dose experiment involved using humanerythropoietin (EPO) mmRNA (mRNA sequence shown in SEQ ID NO: 33900;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1) or G-CSF mmRNA (mRNA sequence shown in SEQ ID NO: 33897;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1) administered in formulation buffer. The dosing vehicle (F.buffer) was used as a control. The EPO and G-CSF modified mRNA weremodified with 5-methylcytosine at each cytosine and pseudouridinereplacement at each uridine site.

Animals (n=5), Sprague-Dawley rats, were injected IM (intramuscular) forthe single unit dose of 100 ug (delivered to one thigh). Formulti-dosing 6 doses of 100 ug (delivered to two thighs) were used forboth EPO and G-CSF mmRNA. Control dosing involved use of buffer at asingle dose. Human EPO blood levels were evaluated 13 hrs postinjection.

Human EPO protein was measured in rat serum 13 hrs post intramuscularinjection. Five groups of rats were treated and evaluated. The resultsare shown in Table 74.

TABLE 74 Multi-dose study Avg. Pg/mL human Dose of Total EPO, GroupTreatment mmRNA Dose serum 1 Human EPO mmRNA 1 × 100 ug 100 ug 143 2Human EPO mmRNA 6 × 100 ug 600 ug 256 3 G-CSF mmRNA 1 × 100 ug 100 ug 43 4 G-CSF mmRNA 6 × 100 ug 600 ug  58 5 Buffer Alone — —  20

Example 55 Signal Sequence Exchange Study

Several variants of mmRNAs encoding human Granulocyte colony stimulatingfactor (G-CSF) (mRNA sequence shown in SEQ ID NO: 33897; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) weresynthesized using modified nucleotides pseudouridine and5-methylcytosine (pseudo-U/5mC). These variants included the G-CSFconstructs encoding either the wild-type N terminal secretory signalpeptide sequence (MAGPATQSPMKLMALQLLLWHSALWTVQEA; SEQ ID NO: 95), nosecretory signal peptide sequence, or secretory signal peptide sequencestaken from other mRNAs. These included sequences where the wild typeG-CSF signal peptide sequence was replaced with the signal peptidesequence of either:

human Factor IX (FIX) SEQ ID NO: 94)human α-1-anti trypsin (AAT) (MMPSSVSWGILLLAGLCCLV PVSLA;, human Prolactin (Prolac) SEQ ID NO: 96)(MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKR;,(MKGSLLLLLVSNLLLCQSVAP; SEQ ID NO: 97),  or human Albumin (Alb)(MKWVTFISLLFLFSSAYSRGVFRR; SEQ ID NO: 98).

250 ng of modified mRNA encoding each G-CSF variant was transfected intoHEK293A (293A in the table), mouse myoblast (MM in the table) (C2C12,CRL-1772, ATCC) and rat myoblast (RM in the table) (L6 line, CRL-1458,ATCC) cell lines in a 24 well plate using 1 ul of Lipofectamine 2000(Life Technologies), each well containing 300,000 cells. Thesupernatants were harvested after 24 hrs and the secreted G-CSF proteinwas analyzed by ELISA using the Human G-CSF ELISA kit (LifeTechnologies). The data shown in Table 75 reveal that cells transfectedwith G-CSF mmRNA encoding the Albumin signal peptide secrete at least 12fold more G-CSF protein than its wild type counterpart.

TABLE 75 Signal Peptide Exchange 293A MM RM Signal peptides (pg/ml)(pg/ml) (pg/ml) G-CSF Natural 9650 3450 6050 α-1-anti trypsin 9950 50008475 Factor IX 11675 6175 11675 Prolactin 7875 1525 9800 Albumin 12205081050 173300 No Signal peptide 0 0 0

Example 56 Cytokine Study: PBMC

A. PBMC isolation and Culture

50 mL of human blood from two donors was received from Research BloodComponents (lots KP30928 and KP30931) in sodium heparin tubes. For eachdonor, the blood was pooled and diluted to 70 mL with DPBS (SAFCBioscience 59331C, lot 071M8408) and split evenly between two 50 mLconical tubes. 10 mL of Ficoll Paque (GE Healthcare 17-5442-03, lot10074400) was gently dispensed below the blood layer. The tubes werecentrifuged at 2000 rpm for 30 minutes with low acceleration andbraking. The tubes were removed and the buffy coat PBMC layers weregently transferred to a fresh 50 mL conical and washed with DPBS. Thetubes were centrifuged at 1450 rpm for 10 minutes.

The supernatant was aspirated and the PBMC pellets were resuspended andwashed in 50 mL of DPBS. The tubes were centrifuged at 1250 rpm for 10minutes. This wash step was repeated, and the PBMC pellets wereresuspended in 19 mL of Optimem I (Gibco 11058, lot 1072088) andcounted. The cell suspensions were adjusted to a concentration of3.0×10̂6 cells/mL live cells.

These cells were then plated on five 96 well tissue culture treatedround bottom plates (Costar 3799) per donor at 50 uL per well. Within 30minutes, transfection mixtures were added to each well at a volume of 50uL per well. After 4 hours post transfection, the media was supplementedwith 10 uL of Fetal Bovine Serum (Gibco 10082, lot 1012368)

B. Transfection Preparation

mmRNA encoding human G-CSF (mRNA sequence shown in SEQ ID NO: 33897;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1) (containing either (1) natural NTPs, (2) 100% substitutionwith 5-methyl cytidine and pseudouridine, or (3) 100% substitution with5-methyl cytidine and N1-methyl pseudouridine; mmRNA encoding luciferase(IVT cDNA sequence shown in SEQ ID NO: 33906; mRNA sequence shown in SEQID NO: 33907, polyA tail of approximately 160 nucleotides not shown insequence, 5′ cap, Cap1, fully modified with 5-methylcytosine at eachcytosine and pseudouridine replacement at each uridine site) (containingeither (1) natural NTPs or (2) 100% substitution with 5-methyl cytidineand pseudouridine) and TLR agonist R848 (Invivogen tlrl-r848) werediluted to 38.4 ng/uL in a final volume of 2500 uL Optimem I.

Separately, 432 uL of Lipofectamine 2000 (Invitrogen 11668-027, lot1070962) was diluted with 13.1 mL Optimem I. In a 96 well plate ninealiquots of 135 uL of each mmRNA, positive control (R-848) or negativecontrol (Optimem I) was added to 135 uL of the diluted Lipofectamine2000. The plate containing the material to be transfected was incubatedfor 20 minutes. The transfection mixtures were then transferred to eachof the human PBMC plates at 50 uL per well. The plates were thenincubated at 37 C. At 2, 4, 8, 20, and 44 hours each plate was removedfrom the incubator, and the supernatants were frozen.

After the last plate was removed, the supernatants were assayed using ahuman G-CSF ELISA kit (Invitrogen KHC2032) and human IFN-alpha ELISA kit(Thermo Scientific 41105-2). Each condition was done in duplicate.

C. Results

The ability of unmodified and modified mRNA (mmRNAs) to produce theencoded protein was assessed (G-CSF production) over time as was theability of the mRNA to trigger innate immune recognition as measured byinterferon-alpha production. Use of in vitro PBMC cultures is anaccepted way to measure the immunostimulatory potential ofoligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102;herein incorporated by reference in its entirety).

Results were interpolated against the standard curve of each ELISA plateusing a four parameter logistic curve fit. Shown in Tables 76 and 77 arethe average from 2 separate PBMC donors of the G-CSF and IFN-alphaproduction over time as measured by specific ELISA.

In the G-CSF ELISA, background signal from the Lipofectamine 2000untreated condition was subtracted at each timepoint. The datademonstrated specific production of human G-CSF protein by humanperipheral blood mononuclear is seen with G-CSF mRNA containing naturalNTPs, 100% substitution with 5-methyl cytidine and pseudouridine, or100% substitution with 5-methyl cytidine and N1-methyl-pseudouridine.Production of G-CSF was significantly increased through the use ofmodified mRNA relative to unmodified mRNA, with the 5-methyl cytidineand N1-methyl-pseudouridine containing G-CSF mmRNA showing the highestlevel of G-CSF production. With regards to innate immune recognition,unmodified mRNA resulted in substantial IFN-alpha production, while themodified mRNA largely prevented interferon-alpha production. G-CSF mRNAfully modified with 5-methyl cytidine and N1-methyl-pseudouridine didnot significantly increase cytokines whereas G-CSF mRNA fully modifiedwith 5-methyl cytidine and pseudouridine induced IFN-alpha, TNF-alphaand IP10. Many other cytokines were not affected by either modification.

TABLE 76 G-CSF Signal G-CSF signal—2 Donor Average p/mL 2 Hr 4 Hr 8 Hr20 Hr 44 Hr G-CSF 120.3 136.8 421.0 346.1 431.8 (5mC/pseudouridine)G-CSF (5mC/N1-methyl 256.3 273.7 919.3 1603.3 1843.3 pseudouridine)G-CSF(Natural-no 63.5 92.6 129.6 258.3 242.4 modification) Luciferase4.5 153.7 33.0 186.5 58.0 (5mC/pseudouridine)

TABLE 77 IFN-alpha signal IFN-alpha signal—2 donor average pg/mL 2 Hr 4Hr 8 Hr 20 Hr 44 Hr G-CSF (5mC/pseudouridine) 21.1 2.9 3.7 22.7 4.3G-CSF (5mC/N1-methyl  0.5 0.4 3.0 2.3 2.1 pseudouridine) G-CSF(Natural) 0.0 2.1 23.3 74.9 119.7 Luciferase  0.4 0.4 4.7 1.0 2.4(5mC/pseudouridine) R-848 39.1 151.3 278.4 362.2 208.1 Lpf. 2000 control 0.8 17.2 16.5 0.7 3.1

Example 57 Chemical Modification Ranges of Modified mRNA

Modified nucleotides such as, but not limited to, the chemicalmodifications 5-methylcytosine and pseudouridine have been shown tolower the innate immune response and increase expression of RNA inmammalian cells. Surprisingly, and not previously known, the effectsmanifested by the chemical modifications can be titrated when the amountof chemical modification is less than 100%. Previously, it was believedthat full modification was necessary and sufficient to elicit thebeneficial effects of the chemical modifications and that less than 100%modification of an mRNA had little effect. However, it has now beenshown that the benefits of chemical modification can be derived usingless than complete modification and that the effects are target,concentration and modification dependent.

A. Modified RNA Transfected in PBMC

960 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with0.8 uL of Lipofectamine 2000 into peripheral blood mononuclear cells(PBMC) from three normal blood donors (D1, D2, D3). The G-CSF mRNA (mRNAsequence shown in SEQ ID NO: 33897; polyA tail of approximately 160nucleotides not shown in sequence; 5′cap, Cap1) was completely modifiedwith 5mC and pseudoU (100% modification), not modified with 5mC andpseudoU (0% modification) or was partially modified with 5mC and pseudoUso the mRNA would contain 50% modification, 25% modification, 10%modification, %5 modification, 1% modification or 0.1% modification. Acontrol sample of Luciferase (mRNA sequence shown in SEQ ID NO: 33907;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified 5meC and pseudoU) was also analyzed forG-CSF expression. For TNF-alpha and IFN-alpha control samples ofLipofectamine 2000, LPS, R-848, Luciferase (mRNA sequence shown in SEQID NO: 33907; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1; fully modified 5mC and pseudo), and P(I)P(C) werealso analyzed. The supernatant was harvested and run by ELISA 22 hoursafter transfection to determine the protein expression. The expressionof G-CSF is shown in Table 78 and the expression of IFN-alpha andTNF-alpha is shown in Table 79. The expression of IFN-alpha andTNF-alpha may be a secondary effect from the transfection of the G-CSFmRNA. Tables 78 and 79 show that the amount of chemical modification ofG-CSF, IFN-alpha and TNF-alpha is titratable when the mRNA is not fullymodified and the titratable trend is not the same for each target.

TABLE 78 G-CSF Expression G-CSF Expression (pg/ml) Dl D2 D3 100%modification 270.3 151.6 162.2 50% modification 45.6 19.8 26.3 25%modification 23.6 10.8 8.9 10% modification 39.4 12.9 12.9 5%modification 70.9 26.8 26.3 1 % modification 70.3 26.9 66.9 0.1%modification 67.5 25.2 28.7 Luciferase 14.5 3.1 10.0

TABLE 79 IFN-alpha and TNF-alpha Expression IFN-alpha ExpressionTNF-alpha Expression (pg/ml) (pg/ml) D1 D2 D3 D1 D2 D3 100% modification76.8 6.8 15.1 5.6 1.4 21.4 50% modification 22.0 5.5 257.3 4.7 1.7 12.125% modification 64.1 14.9 549.7 3.9 0.7 10.1 10% modification 150.218.8 787.8 6.6 0.9 13.4 5% modification 143.9 41.3 1009.6 2.5 1.8 12.01% modification 189.1 40.5 375.2 9.1 1.2 25.7 0.1% modification 261.237.8 392.8 9.0 2. 13.7 0% modification 230.3 45.1 558.3 10.9 1.4 10.9 LF200 0 0 1.5 45.8 2.8 53.6 LPS 0 0 1.0 114.5 70.0 227.0 R-848 39.5 11.9183.5 389.3 256.6 410.6 Luciferase 9.1 0 3.9 4.5 2.7 13.6 P(I)P(C)1498.1 216.8 238.8 61.2 4.4 69.1

B. Modified RNA Transfected in HEK293

Human embryonic kidney epithelial (HEK293) cells were seeded on 96-wellplates at a density of 30,000 cells per well in 100 ul cell culturemedium. 250 ng of modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO:33897; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1) formulated with RNAiMAX™ (Invitrogen, Carlsbad,Calif.) was added to a well. The G-CSF was completely modified with 5mCand pseudoU (100% modification), not modified with 5mC and pseudoU (0%modification) or was partially modified with 5mC and pseudoU so the mRNAwould contain 75% modification, 50% modification or 25% modification.Control samples (AK 5/2, mCherry (SEQ ID NO: 33898; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified 5mC and pseudoU) and untreated) were also analyzed. Thehalf-life of G-CSF mRNA fully modified with 5-methylcytosine andpseudouridine is approximately 8-10 hours. The supernatants wereharvested after 16 hours and the secreted G-CSF protein was analyzed byELISA. Table 80 shows that the amount of chemical modification of G-CSFis titratable when the mRNA is not fully modified.

TABLE 80 G-CSF Expression G-CSF Expression (ng/ml) 100% modification118.4 75% modification 101.9 50% modification 105.7 25% modification231.1 0% modification 270.9 AK 5/2 166.8 mCherry 0 Untreated 0

Example 58 In Vivo Delivery of Modified mRNA (mmRNA)

Modified RNA was delivered to C57/BL6 mice intramuscularly,subcutaneously, or intravenously to evaluate the bio-distribution ofmodified RNA using luciferase. A formulation buffer used with alldelivery methods contained 150 mM sodium chloride, 2 mM calciumchloride, 2 mM Na+-phosphate which included 1.4 mM monobasic sodiumphosphate and 0.6 mM of dibasic sodium phosphate, and 0.5 mMethylenediaminetetraacetic acid (EDTA) was adjusted using sodiumhydroxide to reach a final pH of 6.5 before being filtered andsterilized. A 1× concentration was used as the delivery buffer. Tocreate the lipoplexed solution delivered to the mice, in one vial 50 μgof RNA was equilibrated for 10 minutes at room temperature in thedelivery buffer and in a second vial 10 μl RNAiMAX™ was equilibrated for10 minutes at room temperature in the delivery buffer. Afterequilibrium, the vials were combined and delivery buffer was added toreach a final volume of 100 μl which was then incubated for 20 minutesat room temperature. Luciferin was administered by intraperitonealinjection (IP) at 150 mg/kg to each mouse prior to imaging during theplateau phase of the luciferin exposure curve which was between 15 and30 minutes. To create luciferin, 1 g of D-luciferin potassium or sodiumsalt was dissolved in 66.6 ml of distilled phosphate buffer solution(DPBS), not containing Mg2+ or Ca2+, to make a 15 mg/ml solution. Thesolution was gently mixed and passed through a 0.2 μm syringe filter,before being purged with nitrogen, aliquoted and frozen at −80° C. whilebeing protected from light as much as possible. The solution was thawedusing a waterbath if luciferin was not dissolved, gently mixed and kepton ice on the day of dosing.

Whole body images were taken of each mouse 2, 8 and 24 hours afterdosing. Tissue images and serum was collected from each mouse 24 hoursafter dosing. Mice administered doses intravenously had their liver,spleen, kidneys, lungs, heart, peri-renal adipose tissue and thymusimaged. Mice administered doses intramuscularly or subcutaneously hadtheir liver, spleen, kidneys, lungs, peri-renal adipose tissue, andmuscle at the injection site. From the whole body images thebioluminescence was measured in photon per second for each route ofadministration and dosing regimen.

A. Intramuscular Administration

Mice were intramuscularly (I.M.) administered either modified luciferasemRNA fully modified with 5-methylcytosine and pseudouridine (Naked-Luc),lipoplexed modified luciferase mRNA fully modified with 5-methylcytosineand pseudouridine (Lipoplex-luc) (IVT cDNA sequence shown in SEQ ID NO:33906; mRNA sequence shown in SEQ ID NO: 33907, polyA tail ofapproximately 160 nucleotides not shown in sequence, 5′ cap, Cap1, fullymodified with 5-methylcytosine at each cytosine and pseudouridinereplacement at each uridine site), lipoplexed modified granulocytecolony-stimulating factor (G-CSF) mRNA (mRNA sequence shown in SEQ IDNO: 33897; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) (Lipoplex-Cytokine) or the formation buffer at a singledose of 50 μg of modified RNA in an injection volume of 50 μl for eachformulation in the right hind limb and a single dose of 5 μg of modifiedRNA in an injection volume of 50 μl in the left hind limb. Thebioluminescence average for the luciferase expression signals for eachgroup at 2, 8 and 24 hours after dosing are shown Table 81. Thebioluminescence showed a positive signal at the injection site of the 5μg and 50 μg modified RNA formulations containing and not containinglipoplex.

TABLE 81 In vivo Biophotoic Imaging (I.M. Injection Route) DoseBioluminescence (photon/sec) Formulation (ug) 2 hours 8 hours 24 hoursNaked-Luc 5 224,000 683,000 927,000 Lipolplex-Luc 5 579,000 639,000186,000 Lipoplex-G-CSF 5 64,600 85,600 75,100 Formulation Buffer 5102,000 86,000 90,700 Naked-Luc 50 446,000 766,000 509,000 Lipolplex-Luc50 374,000 501,000 332,000 Lipoplex-G-CSF 50 49,400 74,800 74,200Formulation Buffer 50 59,300 69,200 63,600

B. Subcutaneous Administration

Mice were subcutaneously (S.C.) administered either modified luciferasemRNA (Naked-Luc), lipoplexed modified luciferase mRNA (Lipoplex-luc),lipoplexed modified G-CSF mRNA (Lipoplex-G-CSF) or the formation bufferat a single dose of 50 μg of modified mRNA in an injection volume of 100μl for each formulation. The bioluminescence average for the luciferaseexpression signals for each group at 2, 8 and 24 hours after dosing areshown in Table 82. The bioluminescence showed a positive signal at theinjection site of the 50 μg modified mRNA formulations containing andnot containing lipoplex.

TABLE 82 In vivo Biophotoic Imaging (S.C. Injection Route)Bioluminescence (photon/sec) Formulation 2 hours 8 hours 24 hoursNaked-Luc 3,700,000 8,060,000 2,080,000 Lipolplex-Luc 3,960,0001,700,000 1,290,000 Lipoplex-G-CSF  123,000 121,000 117,000 FormulationBuffer  116,000 127,000 123,000

C. Intravenous Administration

Mice were intravenously (I.V.) administered either modified luciferasemRNA (Naked-Luc), lipoplexed modified luciferase mRNA (Lipoplex-luc),lipoplexed modified G-CSF mRNA (Lipoplex-G-CSF) or the formation bufferat a single dose of 50 μg of modified mRNA in an injection volume of 100μl for each formulation. The bioluminescence average for the luciferaseexpression signal in the spleen from each group at 2 hours after dosingis shown in Table 83. The bioluminescence showed a positive signal inthe spleen of the 50 μg modified mRNA formulations containing lipoplex.

TABLE 83 In vivo Biophotoic Imaging (I.V. InjectionRoute)Bioluminescence (photon/sec) Formulation of the Spleen Naked-Luc 58,400Lipolplex-Luc 65,000 Lipoplex-G-CSF 57,100 Formulation Buffer 58,300

Example 59 Buffer Formulation Studies

G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 33897; polyA tailof approximately 160 nucleotides not shown in sequence; 5′cap, Cap1;fully modified with N1-pseudouridine and 5-methylcytosine) or Factor IXmodified mRNA (mRNA sequence shown in SEQ ID NO: 33901; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with N1-pseudouridine and 5-methylcytosine) in a buffersolution is administered intramuscularly to rats in an injection volumeof 50 μl (n=5) at a modified mRNA dose of 200 ug per rat as described inTable 84. The modified mRNA is lyophilized in water for 1-2 days. It isthen reconstituted in the buffers listed below to a target concentrationof 6 mg/ml. Concentration is determined by OD 260. Samples are dilutedto 4 mg/ml in the appropriate buffer before dosing.

To precipitate the modified mRNA, 3M sodium acetate, pH 5.5 and pureethanol are added at 1/10^(th) the total volume and 4 times the totalvolume of modified mRNA, respectively. The material is placed at −80 Cfor a minimum of 1 hour. The material is then centrifuged for 30 minutesat 4000 rpm, 4 C. The supernatant is removed and the pellet iscentrifuged and washed 3× with 75% ethanol. Finally, the pellet isreconstituted with buffer to a target concentration of 6 mg/ml.Concentration is determined by OD 260. Samples are diluted to 4 mg/ml inthe appropriate buffer before dosing. All samples are prepared bylyophilization unless noted below.

TABLE 84 Buffer Dosing Groups Group Treatment Buffer Dose (ug/rat) 1G-CSF 0.9% Saline 200 Factor IX 0.9% Saline 200 2 G-CSF 0.9% Saline + 2mM 200 Calcium Factor IX 0.9% Saline + 2 mM 200 Calcium 3 G-CSF LactatedRinger's 200 Factor IX Lactated Ringer's 200 4 G-CSF   5% Sucrose 200Factor IX   5% Sucrose 200 5 G-CSF   5% Sucrose + 2 mM 200 CalciumFactor IX   5% Sucrose + 2 mM 200 Calcium 6 G-CSF   5% Mannitol 200Factor IX   5% Mannitol 200 7 G-CSF   5% Mannitol + 2 mM 200 CalciumFactor IX   5% Mannitol + 2 mM 200 Calcium 8 G-CSF 0.9% saline(precipitation) 200 Factor IX 0.9% saline (precipitation) 200

Serum samples are collected from the rats at various time intervals andanalyzed for G-CSF or Factor IX protein expression using G-CSF or FactorIX ELISA.

Example 60 Multi-Dose Study

Sprague-Dawley rats (n=8) are injected intravenously eight times (twicea week) over 28 days. The rats are injected with 0.5 mg/kg, 0.05 mg/kg,0.005 mg/kg or 0.0005 mg/kg of human G-CSF modified mRNA of luciferasemodified mRNA formulated in a lipid nanoparticle, 0.5 mg/kg of humanG-CSF modified mRNA in saline, 0.2 mg/kg of the human G-CSF proteinNeupogen or non-translatable human G-CSF modified mRNA formulated in alipid nanoparticle. Serum is collected during predetermined timeintervals to evaluate G-CSF protein expression (8, 24 and 72 hours afterthe first dose of the week), complete blood count and white blood count(24 and 72 hours after the first dose of the week) and clinicalchemistry (24 and 72 hours after the first dose of the week). The ratsare sacrificed at day 29, 4 days after the final dosing, to determinethe complete blood count, white blood count, clinical chemistry, proteinexpression and to evaluate the effect on the major organs byhistopathology and necropsy. Further, an antibody assay is performed onthe rats on day 29.

Example 61 LNP In Vivo Study

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 160 nucleotides not shown in sequence, 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine wasformulated as a lipid nanoparticle (LNP) using the syringe pump method.The LNP was formulated at a 20:1 weight ratio of total lipid to modifiedmRNA with a final lipid molar ratio of 50:10:38.5:1.5(DLin-KC2-DMA:DSPC:Cholesterol:PEG-DMG). As shown in Table 85, theluciferase LNP formulation was characterized by particle size, zetapotential, and encapsulation.

TABLE 85 Luciferase Formulation Formulation NPA-098-1 Modified mRNALuciferase Mean size 135 nm PDI: 0.08 Zeta at pH 7.4 −0.6 mV Encaps. 91%(RiboGr)

As outlined in Table 86, the luciferase LNP formulation was administeredto Balb-C mice (n=3) intramuscularly, intravenously and subcutaneouslyand a luciferase modified RNA formulated in PBS was administered to miceintravenously.

TABLE 86 Luciferase Formulations Amount Concen- Injection of trationVolume modified Dose Formulation Vehicle Route (mg/ml) (ul) RNA (ug)(mg/kg) Luc-LNP PBS IV 0.20 50 10 0.50 Luc-LNP PBS IM 0.20 50 10 0.50Luc-LNP PBS SC 0.20 50 10 0.50 Luc-PBS PBS IV 0.20 50 10 0.50

The mice administered the luciferase LNP formulation intravenously andintramuscularly were imaged at 2, 8, 24, 48, 120 and 192 hours and themice administered the luciferase LNP formulation subcutaneously wereimaged at 2, 8, 24, 48 and 120 hours to determine the luciferaseexpression as shown in Table 87. In Table 87, “NT” means not tested.Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse.

TABLE 87 Luciferase Expression Average Expression (photon/second) Routeof 2 8 24 48 120 192 Form. Administration hours hours hours hours hourshours Luc- IV 1.62E+08 3.00E+09 7.77E+08 4.98E+08 1.89E+08 6.08E+07 LNPLuc- IM 4.85E+07 4.92E+08 9.02E+07 3.17E+07 1.22E+07 2.38E+06 LNP Luc-SC 1.85E+07 9.79E+08 3.09E+08 4.94E+07 1.98E+06 NT LNP Luc- IV 3.61E+055.64E+05 3.19E+05 NT NT NT PBS

One mouse administered the LNP formulation intravenously was sacrificedat 8 hours to determine the luciferase expression in the liver andspleen. Also, one mouse administered the LNP formulation intramuscularwas sacrificed at 8 hours to determine the luciferase expression of themuscle around the injection site and in the liver and spleen. As shownin Table 88, expression was seen in the both the liver and spleen afterintravenous and intramuscular administration and in the muscle aroundthe intramuscular injection site.

TABLE 88 Luciferase Expression in Tissue Expression (photon/second)Luciferase LNP: IV Administration Liver 7.984E+08 Spleen 3.951E+08Luciferase LNP: IM Administration Muscle around the 3.688E+07 injectionsite Liver 1.507E+08 Spleen 1.096E+07

Example 62 Cytokine Study: PBMC

A. PBMC isolation and Culture

50 mL of human blood from two donors was received from Research BloodComponents (lots KP30928 and KP30931) in sodium heparin tubes. For eachdonor, the blood was pooled and diluted to 70 mL with DPBS (SAFCBioscience 59331C, lot 071M8408) and split evenly between two 50 mLconical tubes. 10 mL of Ficoll Paque (GE Healthcare 17-5442-03, lot10074400) was gently dispensed below the blood layer. The tubes werecentrifuged at 2000 rpm for 30 minutes with low acceleration andbraking. The tubes were removed and the buffy coat PBMC layers weregently transferred to a fresh 50 mL conical and washed with DPBS. Thetubes were centrifuged at 1450 rpm for 10 minutes.

The supernatant was aspirated and the PBMC pellets were resuspended andwashed in 50 mL of DPBS. The tubes were centrifuged at 1250 rpm for 10minutes. This wash step was repeated, and the PBMC pellets wereresuspended in 19 mL of Optimem I (Gibco 11058, lot 1072088) andcounted. The cell suspensions were adjusted to a concentration of3.0×10̂6 cells/mL live cells.

These cells were then plated on five 96 well tissue culture treatedround bottom plates (Costar 3799) per donor at 50 uL per well. Within 30minutes, transfection mixtures were added to each well at a volume of 50uL per well. After 4 hours post transfection, the media was supplementedwith 10 uL of Fetal Bovine Serum (Gibco 10082, lot 1012368).

B. Transfection Preparation

Modified mRNA encoding human G-CSF (mRNA sequence shown in SEQ ID NO:33897; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) (containing either (1) natural NTPs, (2) 100%substitution with 5-methyl cytidine and pseudouridine, or (3) 100%substitution with 5-methyl cytidine and N1-methyl pseudouridine; mRNAencoding luciferase (IVT cDNA sequence shown in SEQ ID NO: 33906; mRNAsequence shown in SEQ ID NO: 33907, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) (containing either (1) natural NTPs or (2) 100%substitution with 5-methyl cytidine and pseudouridine) and TLR agonistR848 (Invivogen tlrl-r848) were diluted to 38.4 ng/uL in a final volumeof 2500 uL Optimem I.

Separately, 110 uL of Lipofectamine 2000 (Invitrogen 11668-027, lot1070962) was diluted with 6.76 mL Optimem I. In a 96 well plate ninealiquots of 135 uL of each mRNA, positive control (R-848) or negativecontrol (Optimem I) was added to 135 uL of the diluted Lipofectamine2000. The plate containing the material to be transfected was incubatedfor 20 minutes. The transfection mixtures were then transferred to eachof the human PBMC plates at 50 uL per well. The plates were thenincubated at 37° C. At 2, 4, 8, 20, and 44 hours each plate was removedfrom the incubator, and the supernatants were frozen.

After the last plate was removed, the supernatants were assayed using ahuman G-CSF ELISA kit (Invitrogen KHC2032) and human IFN-alpha ELISA kit(Thermo Scientific 41105-2). Each condition was done in duplicate.

C. Protein and Innate Immune Response Analysis

The ability of unmodified and modified mRNA to produce the encodedprotein was assessed (G-CSF production) over time as was the ability ofthe mRNA to trigger innate immune recognition as measured byinterferon-alpha production. Use of in vitro PBMC cultures is anaccepted way to measure the immunostimulatory potential ofoligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102).

Results were interpolated against the standard curve of each ELISA plateusing a four parameter logistic curve fit. Shown in Tables 89 and 90 arethe average from 3 separate PBMC donors of the G-CSF, interferon-alpha(IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) production overtime as measured by specific ELISA.

In the G-CSF ELISA, background signal from the Lipofectamine 2000(LF2000) untreated condition was subtracted at each time point. The datademonstrated specific production of human G-CSF protein by humanperipheral blood mononuclear is seen with G-CSF mRNA containing naturalNTPs, 100% substitution with 5-methyl cytidine and pseudouridine, or100% substitution with 5-methyl cytidine and N1-methyl pseudouridine.Production of G-CSF was significantly increased through the use of5-methyl cytidine and N1-methyl pseudouridine modified mRNA relative to5-methyl cytidine and pseudouridine modified mRNA.

With regards to innate immune recognition, while both modified mRNAchemistries largely prevented IFN-alpha and TNF-alpha productionrelative to positive controls (R848, p(I)p(C)), significant differencesdid exist between the chemistries. 5-methyl cytidine and pseudouridinemodified mRNA resulted in low but detectable levels of IFN-alpha andTNF-alpha production, while 5-methyl cytidine and N1-methylpseudouridine modified mRNA resulted in no detectable IFN-alpha andTNF-alpha production.

Consequently, it has been determined that, in addition to the need toreview more than one cytokine marker of the activation of the innateimmune response, it has surprisingly been found that combinations ofmodifications provide differing levels of cellular response (proteinproduction and immune activation). The modification, N1-methylpseudouridine, in this study has been shown to convey added protectionover the standard combination of 5-methylcytidine/pseudouridine exploredby others resulting in twice as much protein and almost 150 foldreduction in immune activation (TNF-alpha).

Given that PBMC contain a large array of innate immune RNA recognitionsensors and are also capable of protein translation, it offers a usefulsystem to test the interdependency of these two pathways. It is knownthat mRNA translation can be negatively affected by activation of suchinnate immune pathways (Kariko et al. Immunity (2005) 23:165-175; Warrenet al. Cell Stem Cell (2010) 7:618-630; each of which is hereinincorporated by reference in its entirety). Using PBMC as an in vitroassay system it is possible to establish a correlation betweentranslation (in this case G-CSF protein production) and cytokineproduction (in this case exemplified by IFN-alpha and TNF-alpha proteinproduction). Better protein production is correlated with lowerinduction of innate immune activation pathway, and new chemistries canbe judged favorably based on this ratio (Table 91).

In this study, the PC Ratio for the two chemical modifications,pseudouridine and N1-methyl pseudouridine, both with 5-methy cytosinewas 4742/141=34 as compared to 9944/1=9944 for the cytokine IFN-alpha.For the cytokine, TNF-alpha, the two chemistries had PC Ratios of 153and 1243, respectively suggesting that for either cytokine, theN1-methylpseudouridine is the superior modification. In Tables 89 and90, “NT” means not tested.

TABLE 89 G-CSF G-CSF: 3 Donor Average (pg/ml) G-CSF 4742 5-methylcytosine/ pseudouridine G-CSF 9944 5 -methylcytosine/N1-methylpseudouridine Luciferase 18 LF2000 16

TABLE 90 IFN-alpha and TNF-alpha IFN-alpha: 3 TNF-alpha: 3 Donor AverageDonor Average (pg/ml) (pg/ml) G-CSF 141 31 5-methyl cytosine/pseudouridine G-CSF 1 8 5-methylcytosine/ N1-methylpseudouridineP(I)P(C) 1104 NT R-848 NT 1477 LF2000 17 25

TABLE 91 G-CSF to Cytokine Ratios G-CSF/IFN-alpha (ratio)G-CSF/TNF-alpha (ratio) 5-methyl 5-methylcytosine/ 5-methyl5-methylcytosine/ cytosine/ N1- cytosine/ N1- pseudouridinemethylpseudouridine pseudouridine methylpseudouridine PC Ratio 34 9944153 1243

Example 63 In Vitro PBMC Studies: Percent Modification

480 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with0.4 uL of Lipofectamine 2000 into peripheral blood mononuclear cells(PBMC) from three normal blood donors (D1, D2, and D3). The G-CSF mRNA(mRNA sequence shown in SEQ ID NO: 33897; polyA tail of approximately160 nucleotides not shown in sequence; 5′cap, Cap1) was completelymodified with 5mC and pseudoU (100% modification), not modified with 5mCand pseudoU (0% modification) or was partially modified with 5mC andpseudoU so the mRNA would contain 75% modification, 50% modification or25% modification. A control sample of Luciferase (mRNA sequence shown inSEQ ID NO: 33907; polyA tail of approximately 160 nucleotides not shownin sequence; 5′cap, Cap1; fully modified 5meC and pseudoU) was alsoanalyzed for G-CSF expression. For TNF-alpha and IFN-alpha controlsamples of Lipofectamine 2000, LPS, R-848, Luciferase (mRNA sequenceshown in SEQ ID NO: 33907; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′cap, Cap1; fully modified 5mC and pseudo), andP(I)P(C) were also analyzed. The supernatant was harvested and run byELISA 22 hours after transfection to determine the protein expression.The expression of G-CSF is shown in Table 94 and the expression ofIFN-alpha and TNF-alpha is shown in Table 95. The expression ofIFN-alpha and TNF-alpha may be a secondary effect from the transfectionof the G-CSF mRNA. Tables 94 and 95 show that the amount of chemicalmodification of G-CSF, interferon alpha (IFN-alpha) and tumor necrosisfactor-alpha (TNF-alpha) is titratable when the mRNA is not fullymodified and the titratable trend is not the same for each target.

By using PBMC as an in vitro assay system it is possible to establish acorrelation between translation (in this case G-CSF protein production)and cytokine production (in this case exemplified by IFN-alpha proteinproduction). Better protein production is correlated with lowerinduction of innate immune activation pathway, and the percentagemodification of a chemistry can be judged favorably based on this ratio(Table 96). As calculated from Tables 94 and 95 and shown in Table 96,full modification with 5-methylcytidine and pseudouridine shows a muchbetter ratio of protein/cytokine production than without anymodification (natural G-CSF mRNA) (100-fold for IFN-alpha and 27-foldfor TNF-alpha). Partial modification shows a linear relationship withincreasingly less modification resulting in a lower protein/cytokineratio.

TABLE 94 G-CSF Expression G-CSF Expression (pg/ml) D1 D2 D3 100%modification 1968.9 2595.6 2835.7  75% modification 566.7 631.4 659.5 50% modification 188.9 187.2 191.9  25% modification 139.3 126.9 102.0 0% modification 194.8 182.0 183.3 Luciferase 90.2 0.0 22.1

TABLE 95 IFN-alpha and TNF-alpha Expression IFN-alpha ExpressionTNF-alpha Expression (pg/ml) (pg/ml) D1 D2 D3 D1 D2 D3 100% modification336.5 78.0 46.4 115.0 15.0 11.1  75% modification 339.6 107.6 160.9107.4 21.7 11.8  50% modification 478.9 261.1 389.7 49.6 24.1 10.4  25%modification 564.3 400.4 670.7 85.6 26.6 19.8  0% modification 1421.6810.5 1260.5 154.6 96.8 45.9 LPS 0.0 0.6 0.0 0.0 12.6 4.3 R-848 0.5 3.014.1 655.2 989.9 420.4 P(I)P(C) 130.8 297.1 585.2 765.8 2362.7 1874.4Lipid only 1952.2 866.6 855.8 248.5 82.0 60.7

TABLE 96 PC Ratio and Effect of Percentage of Modification AverageAverage Average G-CSF/ G-CSF/ % G-CSF IFN-a TNF-a IFN-alpha TNF-alphaModification (pg/ml) (pg/ml) (pg/ml) (PC ratio) (PC ratio) 100 2466 15347 16 52 75 619 202 47 3.1 13 50 189 376 28 0.5 6.8 25 122 545 44 0.22.8 0 186 1164 99 0.16 1.9

Example 64 Modified RNA Transfected in PBMC

500 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with0.4 uL of Lipofectamine 2000 into peripheral blood mononuclear cells(PBMC) from three normal blood donors (D1, D2, and D3). The G-CSF mRNA(mRNA sequence shown in SEQ ID NO: 33897; polyA tail of approximately160 nucleotides not shown in sequence; 5′cap, Cap1) was completelymodified with 5mC and pseudoU (100% modification), not modified with 5mCand pseudoU (0% modification) or was partially modified with 5mC andpseudoU so the mRNA would contain 50% modification, 25% modification,10% modification, %5 modification, 1% modification or 0.1% modification.A control sample of mCherry (mRNA sequence shown in SEQ ID NO: 33898;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified 5meC and pseudouridine), G-CSF fully modifiedwith 5-methylcytosine and pseudouridine (Control G-CSF) and an untreatedcontrol was also analyzed for expression of G-CSF, tumor necrosisfactor-alpha (TNF-alpha) and interferon-alpha (IFN-alpha). Thesupernatant was harvested 6 hours and 18 hours after transfection andrun by ELISA to determine the protein expression. The expression ofG-CSF, IFN-alpha, and TNF-alpha for Donor 1 is shown in Table 97, Donor2 is shown in Table 98 and Donor 3 is shown in Table 99.

Full 100% modification with 5-methylcytidine and pseudouridine resultedin the most protein translation (G-CSF) and the least amount of cytokineproduced across all three human PBMC donors. Decreasing amounts ofmodification results in more cytokine production (IFN-alpha andTNF-alpha), thus further highlighting the importance of fullymodification to reduce cytokines and to improve protein translation (asevidenced here by G-CSF production).

TABLE 97 Donor 1 G-CSF (pg/mL) IFN-alpha (pg/mL) TNF-alpha (pg/mL) 6hours 18 hours 6 hours 18 hours 6 hours 18 hours 100% Mod 1815 2224 1 130 0 75% Mod 591 614 0 89 0 0 50% Mod 172 147 0 193 0 0 25% Mod 111 92 2219 0 0 10% Mod 138 138 7 536 18 0 1% Mod 199 214 9 660 18 3 0.1% Mod222 208 10 597 0 6 0% Mod 273 299 10 501 10 0 Control 957 1274 3 12318633 1620 G-CSF mCherry 0 0 0 10 0 0 Untreated N/A N/A 0 0 1 1

TABLE 98 Donor 2 G-CSF (pg/mL) IFN-alpha (pg/mL) TNF-alpha (pg/mL) 6hours 18 hours 6 hours 18 hours 6 hours 18 hours 100% Mod 2184 2432 0 70 11 75% Mod 935 958 3 130 0 0 50% Mod 192 253 2 625 7 23 25% Mod 153158 7 464 6 6 10% Mod 203 223 25 700 22 39 1% Mod 288 275 27 962 51 660.1% Mod 318 288 33 635 28 5 0% Mod 389 413 26 748 1 253 Control 14611634 1 59 481 814 G-CSF mCherry 0 7 0 1 0 0 Untreated N/A N/A 1 0 0 0

TABLE 99 Donor 3 G-CSF IFN-alpha TNF-alpha (pg/mL) (pg/mL) (pg/mL) 6hours 18 hours 6 hours 18 hours 6 hours 18 hours 100% Mod 6086 7549 7658 11 11 75% Mod 2479 2378 23 752 4 35 50% Mod 667 774 24 896 22 18 25%Mod 480 541 57 1557 43 115 10% Mod 838 956 159 2755 144 123 1% Mod 11081197 235 3415 88 270 0.1% Mod 1338 1177 191 2873 37 363 0% Mod 1463 1666215 3793 74 429 Control 3272 3603 16 1557 731 9066 G-CSF mCherry 0 0 2645 0 0 Untreated N/A N/A 1 1 0 8

Example 65 Innate Immune Response Study in BJ Fibroblasts

A. Single Transfection

Human primary foreskin fibroblasts (BJ fibroblasts) were obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, catalog #30-2003) supplementedwith 10% fetal bovine serum at 37° C., under 5% CO2. BJ fibroblasts wereseeded on a 24-well plate at a density of 300,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 33897; polyA tail of approximately 140 nucleotides notshown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine andpseudouridine (Gen1) or fully modified with 5-methylcytosine andN1-methylpseudouridine (Gen2) having Cap0, Cap1 or no cap wastransfected using Lipofectamine 2000 (Invitrogen, catalog #11668-019),following manufacturer's protocol. Control samples of poly I:C (PIC),Lipofectamine 2000 (Lipo), natural luciferase mRNA (mRNA sequence shownin SEQ ID NO: 33907; polyA tail of approximately 160 nucleotides notshown in sequence; 5′cap, Cap1) and natural G-CSF mRNA were alsotransfected. The cells were harvested after 18 hours, the total RNA wasisolated and DNASE® treated using the RNeasy micro kit (catalog #74004)following the manufacturer's protocol. 100 ng of total RNA was used forcDNA synthesis using High Capacity cDNA Reverse Transcription kit(catalog #4368814) following the manufacturer's protocol. The cDNA wasthen analyzed for the expression of innate immune response genes byquantitative real time PCR using SybrGreen in a Biorad CFX 384instrument following manufacturer's protocol. Table 100 shows theexpression level of innate immune response transcripts relative tohouse-keeping gene HPRT (hypoxanthine phosphoribosytransferase) and isexpressed as fold-induction relative to HPRT. In the table, the panel ofstandard metrics includes: RIG-I is retinoic acid inducible gene 1, IL6is interleukin-6, OAS-1 is oligoadenylate synthetase 1, IFNb isinterferon-beta, AIM2 is absent in melanoma-2, IFIT-1 isinterferon-induced protein with tetratricopeptide repeats 1, PKR isprotein kinase R, TNFa is tumor necrosis factor alpha and IFNa isinterferon alpha.

TABLE 100 Innate Immune Response Transcript Levels Form. RIG-I IL6 OAS-1IFNb AIM2 IFIT-1 PKR TNFa IFNa Natural 71.5 20.6 20.778 11.404 0.251151.218 16.001 0.526 0.067 Luciferase Natural 73.3 47.1 19.359 13.6150.264 142.011 11.667 1.185 0.153 G-CSF PIC 30.0 2.8 8.628 1.523 0.10071.914 10.326 0.264 0.063 G-CSF 0.81 0.22 0.080 0.009 0.008 2.220 1.5920.090 0.027 Gen1-UC G-CSF 0.54 0.26 0.042 0.005 0.008 1.314 1.568 0.0880.038 Gen1- Cap0 G-CSF 0.58 0.30 0.035 0.007 0.006 1.510 1.371 0.0900.040 Gen1- Cap1 G-CSF 0.21 0.20 0.002 0.007 0.007 0.603 0.969 0.1290.005 Gen2-UC G-CSF 0.23 0.21 0.002 0.0014 0.007 0.648 1.547 0.121 0.035Gen2- Cap0 G-CSF 0.27 0.26 0.011 0.004 0.005 0.678 1.557 0.099 0.037Gen2- Cap1 Lipo 0.27 0.53 0.001 0 0.007 0.054 1.536 0.158 0.064

B. Repeat Transfection

Human primary foreskin fibroblasts (BJ fibroblasts) were obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, catalog #30-2003) supplementedwith 10% fetal bovine serum at 37° C., under 5% CO₂. BJ fibroblasts wereseeded on a 24-well plate at a density of 300,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 33897; polyA tail of approximately 140 nucleotides notshown in sequence; 5′cap, Cap1) unmodified, fully modified with5-methylcytosine and pseudouridine (Gen1) or fully modified with5-methylcytosine and N1-methylpseudouridine (Gen2) was transfected dailyfor 5 days following manufacturer's protocol. Control samples ofLipofectamine 2000 (L2000) and mCherry mRNA (mRNA sequence shown in SEQID NO: 33898; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1; fully modified with 5-methylcytidine andpseudouridine) were also transfected daily for 5 days. The results areshown in Table 101.

Unmodified mRNA showed a cytokine response in interferon-beta (IFN-beta)and interleukin-6 (IL-6) after one day. mRNA modified with at leastpseudouridine showed a cytokine response after 2-3 days whereas mRNAmodified with 5-methylcytosine and N1-methylpseudouridine showed areduced response after 3-5 days.

TABLE 101 Cytokine Response Formulation Transfection IFN-beta (pg/ml)IL-6 (pg/ml) G-CSF unmodified 6 hours 0 3596 Day 1 1363 15207 Day 2 23812415 Day 3 225 5017 Day 4 363 4267 Day 5 225 3094 G-CSF Gen 1 6 hours 03396 Day 1 38 3870 Day 2 1125 16341 Day 3 100 25983 Day 4 75 18922 Day 5213 15928 G-CSF Gen 2 6 hours 0 3337 Day 1 0 3733 Day 2 150 974 Day 3213 4972 Day 4 1400 4122 Day 5 350 2906 mCherry 6 hours 0 3278 Day 1 2383893 Day 2 113 1833 Day 3 413 25539 Day 4 413 29233 Day 5 213 20178L2000 6 hours 0 3270 Day 1 13 3933 Day 2 388 567 Day 3 338 1517 Day 4475 1594 Day 5 263 1561

Example 66 In Vivo Detection of Innate Immune Response

In an effort to distinguish the importance of different chemicalmodification of mRNA on in vivo protein production and cytokine responsein vivo, female BALB/C mice (n=5) are injected intramuscularly withG-CSF mRNA (G-CSF mRNA unmod) (mRNA sequence shown in SEQ ID NO: 33897;polyA tail of approximately 160 nucleotides not shown in sequence;) witha 5′ cap of Cap1, G-CSF mRNA fully modified with 5-methylcytosine andpseudouridine (G-CSF mRNA 5mc/pU), G-CSF mRNA fully modified with5-methylcytosine and N1-methylpseudouridine with (G-CSF mRNA 5mc/N1pU)or without a 5′ cap (G-CSF mRNA 5mc/N1 pU no cap) or a control of eitherR848 or 5% sucrose as described in Table 102.

TABLE 102 Dosing Chart Formulation Route Dose (ug/mouse) Dose (ul) G-CSFmRNA unmod I.M. 200 50 G-CSF mRNA 5mc/pU I.M. 200 50 G-CSF mRNA I.M. 20050 5mc/N1pU G-CSF mRNA I.M. 200 50 5mc/N1pU no cap R848 I.M. 75 50 5%sucrose I.M. — 50 Untreated I.M. — —

Blood is collected at 8 hours after dosing. Using ELISA the proteinlevels of G-CSF, TNF-alpha and IFN-alpha is determined by ELISA. 8 hoursafter dosing, muscle is collected from the injection site andquantitative real time polymerase chain reaction (QPCR) is used todetermine the mRNA levels of RIG-I, PKR, AIM-2, IFIT-1, OAS-2, MDA-5,IFN-beta, TNF-alpha, IL-6, G-CSF, CD45 in the muscle.

Example 67 In Vivo Detection of Innate Immune Response Study

Female BALB/C mice (n=5) were injected intramuscularly with G-CSF mRNA(G-CSF mRNA unmod) (mRNA sequence shown in SEQ ID NO: 33897; polyA tailof approximately 160 nucleotides not shown in sequence;) with a 5′cap ofCap1, G-CSF mRNA fully modified with 5-methylcytosine and pseudouridine(G-CSF mRNA 5mc/pU), G-CSF mRNA fully modified with 5-methylcytosine andN1-methylpseudouridine with (G-CSF mRNA 5mc/N1pU) or without a 5′ cap(G-CSF mRNA 5mc/N1 pU no cap) or a control of either R848 or 5% sucroseas described in Table 103. Blood is collected at 8 hours after dosingand using ELISA the protein levels of G-CSF and interferon-alpha(IFN-alpha) is determined by ELISA and are shown in Table 103.

As shown in Table 103, unmodified, 5mc/pU, and 5mc/N1pU modified G-CSFmRNA resulted in human G-CSF expression in mouse serum. The uncapped5mC/N1pU modified G-CSF mRNA showed no human G-CSF expression in serum,highlighting the importance of having a 5′ cap structure for proteintranslation.

As expected, no human G-CSF protein was expressed in the R848, 5%sucrose only, and untreated groups. Importantly, significant differenceswere seen in cytokine production as measured by mouse IFN-alpha in theserum. As expected, unmodified G-CSF mRNA demonstrated a robust cytokineresponse in vivo (greater than the R848 positive control). The 5mc/pUmodified G-CSF mRNA did show a low but detectable cytokine response invivo, while the 5mc/N1pU modified mRNA showed no detectable IFN-alpha inthe serum (and same as vehicle or untreated animals).

Also, the response of 5mc/N1pU modified mRNA was the same regardless ofwhether it was capped or not. These in vivo results reinforce theconclusion that 1) that unmodified mRNA produce a robust innate immuneresponse, 2) that this is reduced, but not abolished, through 100%incorporation of 5mc/pU modification, and 3) that incorporation of5mc/N1pU modifications results in no detectable cytokine response.

Lastly, given that these injections are in 5% sucrose (which has noeffect by itself), these result should accurately reflect theimmunostimulatory potential of these modifications.

From the data it is evident that N1pU modified molecules produce moreprotein while concomitantly having little or no effect on IFN-alphaexpression. It is also evident that capping is required for proteinproduction for this chemical modification. The Protein: Cytokine Ratioof 748 as compared to the PC Ratio for the unmodified mRNA (PC=9) meansthat this chemical modification is far superior as related to theeffects or biological implications associated with IFN-alpha.

TABLE 103 Human G-CSF and Mouse IFN-alpha in serum G-CSF IFN-alpha DoseDose protein expression PC Formulation Route (ug/mouse) (ul) (pg/ml)(pg/ml) Ratio G-CSF mRNA I.M. 200 50 605.6 67.01 9 unmod G-CSF mRNA I.M.200 50 356.5 8.87 40 5mc/pU G-CSF mRNA I.M. 200 50 748.1 0 748 5mc/N1pUG-CSF mRNA IM. 200 50 6.5 0 6.5 5mc/N1pU no cap R848 I.M.  75 50 3.440.97 .08 5% sucrose I.M. — 50 0 1.49 0 Untreated I.M. — — 0 0 0

Example 68 In Vivo Delivery of Modified RNA

Protein production of modified mRNA was evaluated by delivering modifiedG-CSF mRNA or modified Factor IX mRNA to female Sprague Dawley rats(n=6). Rats were injected with 400 ug in 100 ul of G-CSF mRNA (mRNAsequence shown in SEQ ID NO: 33897; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (G-CSF Gen1), G-CSF mRNA fullymodified with 5-methylcytosine and N1-methylpseudouridine (G-CSF Gen2)or Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 33901; polyA tailof approximately 160 nucleotides not shown in sequence; 5′cap, Cap1)fully modified with 5-methylcytosine and pseudouridine (Factor IX Gen1)reconstituted from the lyophilized form in 5% sucrose. Blood wascollected 8 hours after injection and the G-CSF protein level in serumwas measured by ELISA. Table 104 shows the G-CSF protein levels in serumafter 8 hours.

These results demonstrate that both G-CSF Gen 1 and G-CSF Gen 2 modifiedmRNA can produce human G-CSF protein in a rat following a singleintramuscular injection, and that human G-CSF protein production isimproved when using Gen 2 chemistry over Gen 1 chemistry.

TABLE 104 G-CSF Protein in Rat Serum (I.M. Injection Route) FormulationG-CSF protein (pg/ml) G-CSF Gen1 19.37 G-CSF Gen2 64.72 Factor IX Gen 12.25

Example 69 Chemical Modification: In Vitro Studies

A. In Vitro Screening in PBMC

500 ng of G-CSF (mRNA sequence shown in SEQ ID NO: 33897; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) mRNAfully modified with the chemical modification outlined Tables 105 and106 was transfected with 0.4 uL Lipofectamine 2000 into peripheral bloodmononuclear cells (PBMC) from three normal blood donors. Control samplesof LPS, R848, P(I)P(C) and mCherry (mRNA sequence shown in SEQ ID NO:33898; polyA tail of approximately 160 nucleotides not shown insequence, 5′cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) were also analyzed. The supernatant was harvested andstored frozen until analyzed by ELISA to determine the G-CSF proteinexpression, and the induction of the cytokines interferon-alpha (IFN-α)and tumor necrosis factor alpha (TNF-α). The protein expression of G-CSFis shown in Table 105, the expression of IFN-α and TNF-α is shown inTable 106.

The data in Table 105 demonstrates that many, but not all, chemicalmodifications can be used to productively produce human G-CSF in PBMC.Of note, 100% N1-methylpseudouridine substitution demonstrates thehighest level of human G-CSF production (almost 10-fold higher thanpseudouridine itself). When N1-methylpseudouridine is used incombination with 5-methylcytidine a high level of human G-CSF protein isalso produced (this is also higher than when pseudouridine is used incombination with 5 methylcytidine).

Given the inverse relationship between protein production and cytokineproduction in PBMC, a similar trend is also seen in Table 106, where100% substitution with N1-methylpseudouridine results no cytokineinduction (similar to transfection only controls) and pseudouridineshows detectable cytokine induction which is above background.

Other modifications such as N6-methyladenosine and alpha-thiocytidineappear to increase cytokine stimulation.

TABLE 105 Chemical Modifications and G-CSF Protein Expression G-CSFProtein Expression (pg/ml) Donor Donor Donor Chemical Modifications 1 23 Pseudouridine 2477 1,909 1,498 5-methyluridine 318 359 345N1-methyl-pseudouridine 21,495 16,550 12,441 2-thiouridine 932 1,000 6004-thiouridine 5 391 218 5-methoxyuridine 2,964 1,832 1,8005-methylcytosine and pseudouridine (1^(st) set) 2,632 1,955 1,3735-methylcytosine and N1-methyl- 10,232 7,245 6,214 pseudouridine (1^(st)set) 2′Fluoroguanosine 59 186 177 2′Fluorouridine 118 209 1915-methylcytosine and pseudouridine (2^(nd) set) 1,682 1,382 1,0365-methylcytosine and N1-methyl- 9,564 8,509 7,141 pseudouridine (2^(nd)set) 5-bromouridine 314 482 291 5-(2-carbomethoxyvinyl)uridine 77 286177 5-[3(1-E-propenylamino)uridine 541 491 550 α-thiocytidine 105 264245 5-methylcytosine and pseudouridine (3^(rd) set) 1,595 1,432 955N1-methyladenosine 182 177 191 N6-methyladenosine 100 168 2005-methylcytidine 291 277 359 N4-acetylcytidine 50 136 365-formylcytidine 18 205 23 5-methylcytosine and pseudouridine (4^(th)set) 264 350 182 5-methylcytosine and N1-methyl- 9,505 6,927 5,405pseudouridine (4^(th) set) LPS 1,209 786 636 mCherry 5 168 164 R848 709732 636 P(I)P(C) 5 186 182

TABLE 106 Chemical Modifications and Cytokine Expression IFN-αExpression TNF-α Expression (pg/ml) (pg/ml) Chemical Donor Donor DonorDonor Donor Donor Modifications 1 2 3 1 2 3 Pseudouridine 120 77 171 3681 126 5-methyluridine 245 135 334 94 100 157 N1-methyl- 26 75 138 101106 134 pseudouridine 2-thiouridine 100 108 154 133 133 1414-thiouridine 463 258 659 169 126 254 5-methoxyuridine 0 64 133 39 74111 5-methylcytosine and 88 94 148 64 89 121 pseudouridine (1^(st) set)5-methylcytosine and 0 60 136 54 79 126 N1-methyl- pseudouridine (1^(st)set) 2′Fluoroguanosine 107 97 194 91 94 141 2′Fluorouridine 158 103 178164 121 156 5-methylcytosine and 133 92 167 99 111 150 pseudouridine(2^(nd) set) 5-methylcytosine and 0 66 140 54 97 149 N1-methyl-pseudouridine (2^(nd) set) 5-bromouridine 95 86 181 87 106 157 5-(2- 061 130 40 81 116 carbomethoxyvinyl) uridine 5-[3(1-E- 0 58 132 71 90 119propenylamino)uridine α-thiocytidine 1,138 565 695 300 273 2775-methylcytosine and 88 75 150 84 89 130 pseudouridine (3^(rd) set)N1-methyladeno sine 322 255 377 256 157 294 N6-methyladenosine 1,9351,065 1,492 1,080 630 857 5-methylcytidine 643 359 529 176 136 193N4-acetylcytidine 789 593 431 263 67 207 5-formylcytidine 180 93 88 13630 40 5-methylcytosine and 131 28 18 53 24 29 pseudouridine (4^(th) set)5-methylcytosine and 0 0 0 36 14 13 N1-methyl- pseudouridine (4^(th)set) LPS 0 67 146 7,004 3,974 4,020 mCherry 100 75 143 67 100 133 R848674 619 562 11,179 8,546 9,907 P(I)P(C) 470 117 362 249 177 197

B. In Vitro Screening in HeLa Cells

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37 oG in 5% CO₂ atmosphere overnight. Next day, 83 ng ofLuciferase modified RNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) with the chemical modification described in Table 107, werediluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.).

Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 0.2 ul were diluted in 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature.

After 18 to 22 hours of incubation cells expressing luciferase werelysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The lysate volumes were adjusted ordiluted until no more than 2 mio relative light units (RLU) per wellwere detected for the strongest signal producing samples and the RLUsfor each chemistry tested are shown in Table 107. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of theplates without reagent was about 200 relative light units per well.

These results demonstrate that many, but not all, chemical modificationscan be used to productively produce human G-CSF in HeLa cells. Of note,100% N1-methyl-pseudouridine substitution demonstrates the highest levelof human G-CSF production.

TABLE 107 Relative Light Units of Luciferase Chemical Modification RLUN6-methyladenosine (m6a) 534 5-methylcytidine (m5c) 138,428N4-acetylcytidine (ac4c) 235,412 5-formylcytidine (f5c) 4365-methylcytosine/pseudouridine, test A1 48,6595-methylcytosine/N1-methyl-pseudouridine, test A1 190,924 Pseudouridine655,632 1 -methylpseudouridine (m1u) 1,517,998 2-thiouridine (s2u) 33875-methoxyuridine (mo5u) 253,719 5-methylcytosine/pseudouridine , test B1317,744 5-methylcytosine/N1-methyl-pseudouridine, test B1 265,871 5-Bromo-uridine 43,276 5 (2 carbovinyl) uridine 531 5 (3-1E propenyl Amino)uridine 446 5-methylcytosine/p seudouridine , test A2 295,8245-methylcytosine/N1-methyl-pseudouridine, test A2 233,9215-methyluridine 50,932 α-Thio-cytidine 26,3585-methylcytosine/pseudouridine, test B2 481,4775-methylcytosine/N1-methyl-pseudouridine, test B2 271,9895-methylcytosine/pseudouridine, test A3 438,8315-methylcytosine/N1-methyl -pseudouridine, test A3 277,499 UnmodifiedLuciferase 234,802

C. In Vitro Screening in Rabbit Reticulocyte Lysates

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) wasmodified with the chemical modification listed in Table 108 and werediluted in sterile nuclease-free water to a final amount of 250 ng in 10ul. The diluted luciferase was added to 40 ul of freshly prepared RabbitReticulocyte Lysate and the in vitro translation reaction was done in astandard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific,Waltham, Mass.) at 30° C. in a dry heating block. The translation assaywas done with the Rabbit Reticulocyte Lysate (nuclease-treated) kit(Promega, Madison, Wis.) according to the manufacturer's instructions.The reaction buffer was supplemented with a one-to-one blend of providedamino acid stock solutions devoid of either Leucine or Methionineresulting in a reaction mix containing sufficient amounts of both aminoacids to allow effective in vitro translation.

After 60 minutes of incubation, the reaction was stopped by placing thereaction tubes on ice. Aliquots of the in vitro translation reactioncontaining luciferase modified RNA were transferred to white opaquepolystyrene 96-well plates (Corning, Manassas, Va.) and combined with100 ul complete luciferase assay solution (Promega, Madison, Wis.). Thevolumes of the in vitro translation reactions were adjusted or diluteduntil no more than 2 mio relative light units (RLUs) per well weredetected for the strongest signal producing samples and the RLUs foreach chemistry tested are shown in Table 108. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of theplates without reagent was about 200 relative light units per well.

These cell-free translation results very nicely correlate with theprotein production results in HeLa, with the same modificationsgenerally working or not working in both systems. One notable exceptionis 5-formylcytidine modified luciferase mRNA which worked in thecell-free translation system, but not in the HeLa cell-basedtransfection system. A similar difference between the two assays wasalso seen with 5-formylcytidine modified G-CSF mRNA.

TABLE 108 Relative Light Units of Luciferase Chemical Modification RLUN6-methyladenosine (m6a) 398 5-methylcytidine (m5c) 152,989N4-acetylcytidine (ac4c) 60,879 5-formylcytidine (f5c) 55,2085-methylcytosine/pseudouridine, test A1 349,3985-methylcytosine/N1-methyl-pseudouridine, test A1 205,465 Pseudouridine587,795 1-methylp seudouridine (m1u) 589,758 2-thiouridine (s2u) 7085-methoxyuridine (mo5u) 288,647 5-methylcytosine/pseudouridine, test B1454,662 5-methylcytosine/N1-methyl-pseudouridine, test B1 223,732 5-Bromo-uridine 221,879 5 (2 carbovinyl) uridine 225 5 (3-1E propenylAmino) uridine 211 5-methylcytosine/pseudouridine, test A2 558,7795-methylcytosine/N1-methyl-pseudouridine, test A2 333,0825-methyluridine 214,680 α-Thio-cytidine 123,8785-methylcytosine/pseudouridine, test B2 487,8055-methylcytosine/N1-methyl-pseudouridine, test B2 154,0965-methylcytosine/pseudouridine, test A3 413,5355-methylcytosine/N1-methyl-pseudouridine, test A3 292,954 UnmodifiedLuciferase 225,986

Example 70 Chemical Modification: In Vivo Studies

A. In Vivo Screening of G-CSF Modified mRNA

Balb-C mice (n=4) are intramuscularly injected in each leg with modifiedG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 33897; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1), fullymodified with the chemical modifications outlined in Table 109, isformulated in 1×PBS. A control of luciferase modified mRNA (mRNAsequence shown in SEQ ID NO: 33907; polyA tail of approximately 160nucleotides not shown in sequence; 5′cap, Cap1; fully modified withpseudouridine and 5-methylcytosine) and a control of PBS are alsotested. After 8 hours serum is collected to determine G-CSF proteinlevels cytokine levels by ELISA.

TABLE 109 G-CSF mRNA Chemical Modifications G-CSF Pseudouridine G-CSF5-methyluridine G-CSF 2-thiouridine G-CSF 4-thiouridine G-CSF5-methoxyuridine G-CSF 2′-fluorouridine G-CSF 5-bromouridine G-CSF5-[3(1-E-propenylamino)uridine) G-CSF alpha-thio-cytidine G-CSF5-methylcytidine G-CSF N4-acetylcytidine G-CSF Pseudouridine and5-methylcytosine G-CSF N1-methyl-pseudouridine and 5-methylcytosineLuciferase Pseudouridine and 5-methylcytosine PBS None

B. In Vivo Screening of Luciferase Modified mRNA

Balb-C mice (n=4) were subcutaneously injected with 200 ul containing 42to 103 ug of modified luciferase mRNA (mRNA sequence shown in SEQ ID NO:33907; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1), fully modified with the chemical modificationsoutlined in Table 110, was formulated in 1×PBS. A control of PBS wasalso tested. The dosages of the modified luciferase mRNA is alsooutlined in Table 110. 8 hours after dosing the mice were imaged todetermine luciferase expression. Twenty minutes prior to imaging, micewere injected intraperitoneally with a D-luciferin solution at 150mg/kg. Animals were then anesthetized and images were acquired with anIVIS Lumina II imaging system (Perkin Elmer). Bioluminescence wasmeasured as total flux (photons/second) of the entire mouse.

As demonstrated in Table 110, all luciferase mRNA modified chemistriesdemonstrated in vivo activity, with the exception of 2′-fluorouridine.In addition 1-methylpseudouridine modified mRNA demonstrated very highexpression of luciferase (5-fold greater expression than pseudouridinecontaining mRNA).

TABLE 110 Luciferase Screening Luciferase Dose Dose expression Chemical(ug) volume (photon/ mRNA Modifications of mRNA (ml) second) Luciferase5-methylcytidine 83 0.72 1.94E+07 Luciferase N4-acetylcytidine 76 0.721.11E07 Luciferase Pseudouridine 95 1.20 1.36E+07 Luciferase1-methylpseudouridine 103 0.72 7.40E+07 Luciferase 5-methoxyuridine 951.22 3.32+07 Luciferase 5-methyluridine 94 0.86 7.42E+06 Luciferase5-bromouridine 89 1.49 3.75E+07 Luciferase 2’-fluoroguanosine 42 0.725.88E+05 Luciferase 2’-fluorocytidine 47 0.72 4.21E+05 Luciferase2’-flurorouridine 59 0.72 3.47E+05 PBS None — 0.72 3.16E+05

Example 71 In Vivo Screening of Combination Luciferase Modified mRNA

Balb-C mice (n=4) were subcutaneously injected with 200 ul of 100 ug ofmodified luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1), fully modified with the chemical modifications outlined in Table111, was formulated in 1×PBS. A control of PBS was also tested. 8 hoursafter dosing the mice were imaged to determine luciferase expression.Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse.

As demonstrated in Table 111, all luciferase mRNA modified chemistries(in combination) demonstrated in vivo activity. In addition the presenceof N1-methyl-pseudouridine in the modified mRNA (with N4-acetylcytidineor 5 methylcytidine) demonstrated higher expression than when the samecombinations where tested using with pseudouridine. Taken together,these data demonstrate that N1-methyl-pseudouridine containingluciferase mRNA results in improved protein expression in vivo whetherused alone (Table 110) or when used in combination with other modifiednucleotides (Table 111).

TABLE 111 Luciferase Screening Combinations Luciferase expression(photon/ mRNA Chemical Modifications second) LuciferaseN4-acetylcytidine/pseudouridine 4.18E+06 LuciferaseN4-acetylcytidine/N1-methyl-pseudouridine 2.88E+07 Luciferase5-methylcytidine/5-methoxyuridine 3.48E+07 Luciferase5-methylcytidine/5-methyluridine 1.44E+07 Luciferase5-methylcytidine/where 50% of the uridine is 2.39E+06 replaced with2-thiouridine Luciferase 5-methylcytidine/pseudouridine 2.36E+07Luciferase 5-methylcytidine/N1-methyl-pseudouridine 4.15E+07 PBS None3.59E+05

Example 72 Innate Immune Response in BJ Fibroblasts

Human primary foreskin fibroblasts (BJ fibroblasts) are obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, cat #30-2003) supplemented with10% fetal bovine serum at 37° C., under 5% CO₂. BJ fibroblasts areseeded on a 24-well plate at a density of 130,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 33897; polyA tail of approximately 160 nucleotides notshown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine andpseudouridine (Gen1) or fully modified with 5-methylcytosine andN1-methylpseudouridine (Gen2) is transfected using Lipofectamine 2000(Invitrogen, cat #11668-019), following manufacturer's protocol. Controlsamples of Lipofectamine 2000 and unmodified G-CSF mRNA (natural G-CSF)are also transfected. The cells are transfected for five consecutivedays. The transfection complexes are removed four hours after each roundof transfection.

The culture supernatant is assayed for secreted G-CSF (R&D Systems,catalog #DCS50), tumor necrosis factor-alpha (TNF-alpha) and interferonalpha (IFN-alpha) by ELISA every day after transfection followingmanufacturer's protocols. The cells are analyzed for viability usingCELL TITER GLO® (Promega, catalog #G7570) 6 hrs and 18 hrs after thefirst round of transfection and every alternate day following that. Atthe same time from the harvested cells, total RNA is isolated andtreated with DNASE® using the RNAEASY micro kit (catalog #74004)following the manufacturer's protocol. 100 ng of total RNA is used forcDNA synthesis using the High Capacity cDNA Reverse Transcription kit(Applied Biosystems, cat #4368814) following the manufacturer'sprotocol. The cDNA is then analyzed for the expression of innate immuneresponse genes by quantitative real time PCR using SybrGreen in a BioradCFX 384 instrument following the manufacturer's protocol.

Example 73 In Vitro Transcription with Wild-Type T7 Polymerase

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 33897; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) werefully modified with different chemistries and chemistry combinationslisted in Tables 112-115 using wild-type T7 polymerase as previouslydescribed.

The yield of the translation reactions was determined byspectrophometric measurement (OD260) and the yield for Luciferase isshown in Table 112 and G-CSF is shown in Table 114. The luciferase andG-CSF modified mRNA were also subjected to an enzymatic capping reactionand each modified mRNA capping reaction was evaluated for yield byspectrophometic measurement (OD260) and correct size assessed usingbioanalyzer. The yield from the capping reaction for luciferase is shownin Table 113 and G-CSF is shown in Table 115.

TABLE 112 In vitro transcription chemistry for Luciferase Yield ChemicalModification (mg) N6-methyladenosine 0.99 5-methylcytidine 1.29N4-acetylcytidine 1.0 5-formylcytidine 0.55 Pseudouridine 2.0N1-methyl-pseudouridine 1.43 2-thiouridine 1.56 5-methoxyuridine 2.355-methyluridine 1.01 α-Thio-cytidine 0.83 5-Br-uridine (5Bru) 1.96 5 (2carbomethoxyvinyl) uridine 0.89 5 (3-1E propenyl Amino) uridine 2.01N4-acetylcytidine/pseudouridine 1.34N4-acetylcytidine/N1-methyl-pseudouridine 1.265-methylcytidine/5-methoxyuridine 1.38 5-methylcytidine/5-bromouridine0.12 5-methylcytidine/5-methyluridine 2.97 5-methylcytidine/half of theuridines 1.59 are modified with 2-thiouridine5-methylcytidine/2-thiouridine 0.90 5-methylcytidine/pseudouridine 1.835-methylcytidine/N1-methyl-pseudouridine 1.33

TABLE 113 Capping chemistry and yield for Luciferase modified mRNA YieldChemical Modification (mg) 5-methylcytidine 1.02 N4-acetylcytidine 0.935-formylcytidine 0.55 Pseudouridine 2.07 N1-methyl-pseudouridine 1.272-thiouridine 1.44 5-methoxyuridine 2 5-methyluridine 0.8α-Thio-cytidine 0.74 5-Br-uridine (5Bru) 1.29 5 (2 carbomethoxyvinyl)uridine 0.54 5 (3-1E propenyl Amino) uridine 1.39N4-acetylcytidine/pseudouridine 0.99N4-acetylcytidine/N1-methyl-pseudouridine 1.085-methylcytidine/5-methoxyuridine 1.13 5-methylcytidine/5-methyluridine1.08 5-methylcytidine/half of the uridines 1.2 are modified with2-thiouridine 5-methylcytidine/2-thiouridine 1.275-methylcytidine/pseudouridine 1.195-methylcytidine/N1-methyl-pseudouridine 1.04

TABLE 114 In vitro transcription chemistry and yield for G-CSF modifiedmRNA Yield Chemical Modification (mg) N6-methyladenosine 1.575-methylcytidine 2.05 N4-acetylcytidine 3.13 5-formylcytidine 1.41Pseudouridine 4.1 N1-methyl-pseudouridine 3.24 2-thiouridine 3.46 5-methoxyuridine 2.57 5-methyluridine 4.27 4-thiouridine 1.452′-F-uridine 0.96 α-Thio-cytidine 2.29 2′-F-guanosine 0.6N-1-methyladenosine 0.63 5-Br-uridine (5Bru) 1.08 5 (2carbomethoxyvinyl) uridine 1.8 5 (3-1E propenyl Amino) uridine 2.09N4-acetylcytidine/p seudouridine 1.72N4-acetylcytidine/N1-methyl-pseudouridine 1.375-methylcytidine/5-methoxyuridine 1.85 5-methylcytidine/5-methyluridine1.56 5-methylcytidine/half of the uridines 1.84 are modified with2-thiouridine 5-methylcytidine/2-thiouridine 2.535-methylcytidine/pseudouridine 0.63 N4-acetylcytidine/2-thiouridine 1.3N4-acetylcytidine/5-bromouridine 1.375-methylcytidine/N1-methyl-pseudouridine 1.25N4-acetylcytidine/pseudouridine 2.24

TABLE 115 Capping chemistry and yield for G-CSF modified mRNA YieldChemical Modification (mg) N6-methyladenosine 1.04 5-methylcytidine 1.08N4-acetylcytidine 2.73 5-formylcytidine 0.95 Pseudouridine 3.88N1-methyl-pseudouridine 2 .58 2-thiouridine 2.57 5-methoxyuridine 2.055-methyluridine 3.56 4-thiouridine 0.91 2′-F-uridine 0.54α-Thio-cytidine 1.79 2′-F-guanosine 0.14 5-Br-uridine (5Bru) 0.79 5 (2carbomethoxyvinyl) uridine 1.28 5 (3-1E propenyl Amino) uridine 1.78N4-acetylcytidine/pseudouridine 0.29N4-acetylcytidine/N1-methyl-pseudouridine 0.335-methylcytidine/5-methoxyuridine 0.91 5-methylcytidine/5-methyluridine0.61 5-methylcytidine/half of the uridines are 1.24 modified with2-thiouridine 5 -methylcytidine/pseudouridine 1.08N4-acetylcytidine/2-thiouridine 1.34 N4-acetylcytidine/5-bromouridine1.22 5-methylcytidine/N1-methyl-pseudouridine 1.56

Example 74 In Vitro Transcription with Mutant T7 Polymerase

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 33897; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) werefully modified with different chemistries and chemistry combinationslisted in Tables 116-119 using a mutant T7 polymerase (Durascribe® T7Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.).

The yield of the translation reactions was determined byspectrophometric measurement (OD260) and the yield for Luciferase isshown in Table 116 and G-CSF is shown in Table 118.

The luciferase and G-CSF modified mRNA were also subjected to anenzymatic capping reaction and each modified mRNA capping reaction wasevaluated for yield by spectrophometic measurement (OD260) and correctsize assessed using bioanalyzer. The yield from the capping reaction forluciferase is shown in Table 117 and G-CSF is shown in Table 119.

TABLE 116 In vitro transcription chemistry and yield for Luciferasemodified mRNA Yield Chemical Modification (ug) 2’Fluorocytosine 71.42’Fluorouridine 57.5 5-methylcytosine/pseudouridine, test A 26.45-methylcytosine/N1-methyl-pseudouridine, test A 73.3N1-acetylcytidine/2-fluorouridine 202.2 5-methylcytidine/2-fluorouridine131.9 2-fluorocytosine/pseudouridine 119.32-fluorocytosine/N1-methyl-pseudouridine 107.02-fluorocytosine/2-thiouridine 34.7 2-fluorocytosine/5-bromouridine 81.02-fluorocytosine/2-fluorouridine 80.4 2-fluoroguanine/5-methylcytosine61.2 2-fluoroguanine/5-methylcytosine/pseudouridine 65.02-fluoroguanine/5-methylcytidine/ 41.2 N1-methyl-pseudouridine2-fluoroguanine/pseudouridine 79.12-fluoroguanine/N1-methyl-pseudouridine 74.65-methylcytidine/pseudouridine, test B 91.85-methylcytidine/N1-methyl-pseudouridine, test B 72.4 2’fluoroadenosine190.98

TABLE 117 Capping chemistry and yield for Luciferase modified mRNA YieldChemical Modification (ug) 2’Fluorocytosine 19.2 2’Fluorouridine 16.75-methylcytosine/pseudouridine, test A 7.05-methylcytosine/N1-methyl-pseudouridine, test A 21.5N1-acetylcytidine/2-fluorouridine 47.5 5-methylcytidine/2-fluorouridine53.2 2-fluorocytosine/pseudouridine 58.42-fluorocytosine/N1-methyl-pseudouridine 26.22-fluorocytosine/2-thiouridine 12.9 2-fluorocytosine/5-bromouridine 26.52-fluorocytosine/2-fluorouridine 35.7 2-fluoroguanine/5-methylcytosine24.7 2-fluoroguanine/5-methylcytosine/pseudouridine 32.32-fluoroguanine/5-methylcytidine/ 31.3 N1-methyl-pseudouridine2-fluoroguanine/pseudouridine 20.92-fluoroguanine/N1-methyl-pseudouridine 29.85-methylcytidine/pseudouridine, test B 58.25-methylcytidine/N1-methyl-pseudouridine, test B 44.4

TABLE 118 In vitro transcription chemistry and yield for G-CSF modifiedmRNA Yield Chemical Modification (ug) 2’Fluorocytosine 56.52’Fluorouridine 79.4 5-methylcytosine/pseudouridine, test A 21.25-methylcytosine/N1-methyl-pseudouridine, test A 77.1N1-acetylcytidine/2-fluorouridine 168.6 5-methylcytidine/2-fluorouridine134.7 2-fluorocytosine/pseudouridine 97.82-fluorocytosine/N1-methyl-pseudouridine 103.12-fluorocytosine/2-thiouridine 58.8 2-fluorocytosine/5-bromouridine 88.82-fluorocytosine/2-fluorouridine 93.9 2-fluoroguanine/5-methylcytosine97.3 2-fluoroguanine/5-methylcytosine/pseudouridine 96.02-fluoroguanine/5-methylcytidine/ 82.0 N1-methyl-pseudouridine2-fluoroguanine/pseudouridine 68.02-fluoroguanine/N1-methyl-pseudouridine 59.35-methylcytidine/pseudouridine, test B 58.75-methylcytidine/N1-methyl-pseudouridine, test B 78.0

TABLE 119 Capping chemistry and yield for G-CSF modified mRNA YieldChemical Modification (ug) 2’Fluorocytosine 16.9 2’Fluorouridine 17.05-methylcytosine/pseudouridine, test A 10.65-methylcytosine/N1-methyl-pseudouridine, test A 22.7N1-acetylcytidine/2-fluorouridine 19.9 5-methylcytidine/2-fluorouridine21.3 2-fluorocytosine/pseudouridine 65.22-fluorocytosine/N1-methyl-pseudouridine 58.92-fluorocytosine/2-thiouridine 41.2 2-fluorocytosine/5-bromouridine 35.82-fluorocytosine/2-fluorouridine 36.7 2-fluoroguanine/5-methylcytosine36.6 2-fluoroguanine/5-methylcytosine/pseudouridine 37.32-fluoroguanine/5-methylcytidine/ 30.7 N1-methyl-pseudouridine2-fluoroguanine/pseudouridine 29.02-fluoroguanine/N1-methyl-pseudouridine 22.75-methylcytidine/pseudouridine, test B 60.45-methylcytidine/N1-methyl-pseudouridine, test B 33.0

Example 75 2′O-methyl and 2′Fluoro compounds

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) wereproduced as fully modified versions with the chemistries in Table 120and transcribed using mutant T7 polymerase (Durascribe® T7 Transcriptionkit (Cat. No. DS010925) (Epicentre®, Madison, Wis.). 2′fluoro-containing mRNA were made using Durascribe T7, however,2′Omethyl-containing mRNA could not be transcribed using Durascribe T7.

Incorporation of 2′Omethyl modified mRNA might possibly be accomplishedusing other mutant T7 polymerases (Nat Biotechnol. (2004) 22:1155-1160;Nucleic Acids Res. (2002) 30:e138) or U.S. Pat. No. 7,309,570, thecontents of each of which are incorporated herein by reference in theirentirety. Alternatively, 2′OMe modifications could be introducedpost-transcriptionally using enzymatic means.

Introduction of modifications on the 2′ group of the sugar has manypotential advantages. 2′OMe substitutions, like 2′ fluoro substitutionsare known to protect against nucleases and also have been shown toabolish innate immune recognition when incorporated into other nucleicacids such as siRNA and anti-sense (incorporated in its entirety,Crooke, ed. Antisense Drug Technology, 2^(nd) edition; Boca Raton: CRCpress).

The 2′Fluoro-modified mRNA were then transfected into HeLa cells toassess protein production in a cell context and the same mRNA were alsoassessed in a cell-free rabbit reticulocyte system. A control ofunmodified luciferase (natural luciferase) was used for bothtranscription experiments, a control of untreated and mock transfected(Lipofectamine 2000 alone) were also analyzed for the HeLa transfectionand a control of no RNA was analyzed for the rabbit reticulysates.

For the HeLa transfection experiments, the day before transfection,20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested bytreatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island,N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplementedwith 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate(Corning, Manassas, Va.). The cells were grown at 37 oG in 5% CO₂atmosphere overnight. Next day, 83 ng of the 2′fluoro-containingluciferase modified RNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) with the chemical modification described in Table 120, werediluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul were diluted in 10 ul finalvolume of OPTI-MEM. After 5 minutes of incubation at room temperature,both solutions were combined and incubated an additional 15 minute atroom temperature. Then the 20 ul combined solution was added to the 100ul cell culture medium containing the HeLa cells and incubated at roomtemperature. After 18 to 22 hours of incubation cells expressingluciferase were lysed with 100 ul of Passive Lysis Buffer (Promega,Madison, Wis.) according to manufacturer instructions. Aliquots of thelysates were transferred to white opaque polystyrene 96-well plates(Corning, Manassas, Va.) and combined with 100 ul complete luciferaseassay solution (Promega, Madison, Wis.). The lysate volumes wereadjusted or diluted until no more than 2 mio relative light units (RLU)per well were detected for the strongest signal producing samples andthe RLUs for each chemistry tested are shown in Table 120. The platereader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The backgroundsignal of the plates without reagent was about 200 relative light unitsper well.

For the rabbit reticulocyte lysate assay, 2′-fluoro-containingluciferase mRNA were diluted in sterile nuclease-free water to a finalamount of 250 ng in 10 ul and added to 40 ul of freshly prepared RabbitReticulocyte Lysate and the in vitro translation reaction was done in astandard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific,Waltham, Mass.) at 30° C. in a dry heating block. The translation assaywas done with the Rabbit Reticulocyte Lysate (nuclease-treated) kit(Promega, Madison, Wis.) according to the manufacturer's instructions.The reaction buffer was supplemented with a one-to-one blend of providedamino acid stock solutions devoid of either Leucine or Methionineresulting in a reaction mix containing sufficient amounts of both aminoacids to allow effective in vitro translation. After 60 minutes ofincubation, the reaction was stopped by placing the reaction tubes onice.

Aliquots of the in vitro translation reaction containing luciferasemodified RNA were transferred to white opaque polystyrene 96-well plates(Corning, Manassas, Va.) and combined with 100 ul complete luciferaseassay solution (Promega, Madison, Wis.). The volumes of the in vitrotranslation reactions were adjusted or diluted until no more than 2 miorelative light units (RLUs) per well were detected for the strongestsignal producing samples and the RLUs for each chemistry tested areshown in Table 121. The plate reader was a BioTek Synergy H1 (BioTek,Winooski, Vt.). The background signal of the plates without reagent wasabout 160 relative light units per well.

As can be seen in Table 120 and 121, multiple 2′Fluoro-containingcompounds are active in vitro and produce luciferase protein.

TABLE 120 HeLa Cells Chemical Concentration Volume Yield Modification(ug/ml) (ul) (ug) RLU 2’Fluoroadenosine 381.96 500 190.98 388.52’Fluorocytosine 654.56 500 327.28 2420 2’Fluoroguanine 541.795 500270.90 11,705.5 2’Flurorouridine 944.005 500 472.00 6767.5 Naturalluciferase N/A N/A N/A 133,853.5 Mock N/A N/A N/A 340 Untreated N/A N/AN/A 238

TABLE 121 Rabbit Reticulysates Chemical Modification RLU2’Fluoroadenosine 162 2’Fluorocytosine 208 2’Fluoroguanine 371,5092’Flurorouridine 258 Natural luciferase 2,159,968 No RNA 156

Example 76 Luciferase in HeLa Cells Using a Combination of Modifications

To evaluate using of 2′fluoro-modified mRNA in combination with othermodification a series of mRNA were transcribed using either wild-type T7polymerase (non-fluoro-containing compounds) or using mutant T7polymerases (fluyoro-containing compounds) as described in Example 75.All modified mRNA were tested by in vitro transfection in HeLa cells.

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37 oG in 5% CO₂ atmosphere overnight. Next day, 83 ng ofLuciferase modified RNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) with the chemical modification described in Table 122, werediluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.).

Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 0.2 ul were diluted in 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature.

After 18 to 22 hours of incubation cells expressing luciferase werelysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The lysate volumes were adjusted ordiluted until no more than 2 mio relative light units (RLU) per wellwere detected for the strongest signal producing samples and the RLUsfor each chemistry tested are shown in Table 122. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of theplates without reagent was about 200 relative light units per well.

As evidenced in Table 122, most combinations of modifications resultedin mRNA which produced functional luciferase protein, including all thenon-fluoro containing compounds and many of the combinations containing2′fluoro modifications.

TABLE 122 Luciferase Chemical Modification RLUN4-acetylcytidine/pseudouridine 113,796N4-acetylcytidine/N1-methyl-pseudouridine 316,3265-methylcytidine/5-methoxyuridine 24,9485-methylcytidine/5-methyluridine 43,675 5-methylcytidine/half of theuridines 41,601 modified with 50% 2-thiouridine5-methylcytidine/2-thiouridine 1,102 5-methylcytidine/pseudouridine51,035 5-methylcytidine/N1-methyl-pseudouridine 152,151N4-acetylcytidine/2’Fluorouridine triphosphate 2885-methylcytidine/2’Fluorouridine triphosphate 269 2’Fluorocytosinetriphosphate/pseudouridine 260 2’Fluorocytosinetriphosphate/N1-methyl-pseudouridine 412 2’Fluorocytosinetriphosphate/2-thiouridine 427 2’Fluorocytosinetriphosphate/5-bromouridine 253 2’Fluorocytosinetriphosphate/2’Fluorouridine triphosphate 184 2’Fluoroguaninetriphosphate/5-methylcytidine 321 2’Fluoroguaninetriphosphate/5-methylcytidine/Pseudouridine 2072’Fluoroguanine/5-methylcytidine/N1-methyl-psuedouridine 2352’Fluoroguanine/pseudouridine 2182’Fluoroguanine/N1-methyl-psuedouridine 2475-methylcytidine/pseudouridine, test A 13,8335-methylcytidine/N1-methyl-pseudouridine, test A 598 2’Fluorocytosinetriphosphate 201 2’Fluorouridine triphosphate 3055-methylcytidine/pseudouridine, test B 115,4015-methylcytidine/N1-methyl-pseudouridine, test B 21,034 Naturalluciferase 30,801 Untreated 344 Mock 262

Example 77 G-CSF In Vitro Transcription

To assess the activity of all our different chemical modifications inthe context of a second open reading frame, we replicated experimentspreviously conducted using luciferase mRNA, with human G-CSF mRNA. G-CSFmRNA (mRNA sequence shown in SEQ ID NO: 33897; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) werefully modified with the chemistries in Tables 124 and 125 usingwild-type T7 polymerase (for all non-fluoro-containing compounds) ormutant T7 polymerase (for all fluoro-containing compounds). The mutantT7 polymerase was obtained commercially (Durascribe® T7 Transcriptionkit (Cat. No. DS010925) (Epicentre®, Madison, Wis.).

The modified RNA in Tables 124 and 125 were transfected in vitro in HeLacells or added to rabbit reticulysates (250 ng of modified mRNA) asindicated. A control of untreated, mock transfected (transfectionreagent alone), G-CSF fully modified with 5-methylcytosine andN1-methyl-pseudouridine or luciferase control (mRNA sequence shown inSEQ ID NO: 33907; polyA tail of approximately 160 nucleotides not shownin sequence; 5′cap, Cap1) fully modified with 5-methylcytosine andN1-methylpseudouridine were also analyzed. The expression of G-CSFprotein was determined by ELISA and the values are shown in Tables 124and 125. In Table 124, “NT” means not tested.

As shown in Table 124, many, but not all, chemical modificationsresulted in human G-CSF protein production. These results fromcell-based and cell-free translation systems correlate very nicely withthe same modifications generally working or not working in both systems.One notable exception is 5-formylcytidine modified G-CSF mRNA whichworked in the cell-free translation system, but not in the HeLacell-based transfection system. A similar difference between the twoassays was also seen with 5-formylcytidine modified luciferase mRNA.

As demonstrated in Table 125, many, but not all, G-CSF mRNA modifiedchemistries (when used in combination) demonstrated in vivo activity. Inaddition the presence of N1-methyl-pseudouridine in the modified mRNA(with N4-acetylcytidine or 5 methylcytidine) demonstrated higherexpression than when the same combinations where tested using withpseudouridine. Taken together, these data demonstrate thatN1-methyl-pseudouridine containing G-CSF mRNA results in improvedprotein expression in vitro.

TABLE 124 G-CSF Expression G-CSF protein G-CSF (pg/ml) protein Rabbit(pg/ml) reticulysates Chemical Modification HeLa cells cellsPseudouridine 1,150,909 147,875 5-methyluridine 347,045 147,2502-thiouridine 417,273 18,375 N1-methylpseudouridine NT 230,0004-thiouridine 107,273 52,375 5-methoxyuridine 1,715,909 201,7505-methylcytosine/pseudouridine, Test A 609,545 119,7505-methylcytosine/N1-methyl- 1,534,318 110,500 pseudouridine, Test A2’-Fluoro-guanosine 11,818 0 2’-Fluoro-uridine 60,455 05-methylcytosine/pseudouridine, Test B 358,182 57,8755-methylcytosine/N1-methyl- 1,568,636 76,750 pseudouridine, Test B5-Bromo-uridine 186,591 72,000 5-(2carbomethoxyvinyl) uridine 1,364 05-[3(1-E-propenylamino) uridine 27,955 32,625 α-thio-cytidine 120,45542,625 5-methylcytosine/pseudouridine, Test C 882,500 49,250N1-methyl-adenosine 4,773 0 N6-methyl-adenosine 1,591 05-methyl-cytidine 646,591 79,375 N4-acetylcytidine 39,545 8,0005-formyl-cytidine 0 24,000 5-methylcytosine/pseudouridine, Test D 87,04547,750 5-methylcytosine/N1-methyl- 1,168,864 97,125 pseudouridine, TestD Mock 909 682 Untreated 0 0 5-methylcytosine/N1-methyl- 1,106,591 NTpseudouridine, Control Luciferase control NT 0

TABLE 125 Combination Chemistries in HeLa cells G-CSF protein (pg/ml)Chemical Modification HeLa cells N4-acetylcytidine/pseudouridine 537,273N4-acetylcytidine/N1-methyl-pseudouridine 1,091,8185-methylcytidine/5-methoxyuridine 516,1365-methylcytidine/5-bromouridine 48,864 5-methylcytidine/5-methyluridine207,500 5-methylcytidine/2-thiouridine 33,409N4-acetylcytidine/5-bromouridine 211,591 N4-acetylcytidine/2-thiouridine46,136 5-methylcytosine/pseudouridine 301,3645-methylcytosine/N1-methyl-pseudouridine 1,017,727N4-acetylcytidine/2’Fluorouridine triphosphate 62,2735-methylcytidine/2’Fluorouridine triphosphate 49,318 2’Fluorocytosinetriphosphate/pseudouridine 7,955 2’Fluorocytosine triphosphate/ 1,364N1-methyl-pseudouridine 2’Fluorocytosine triphosphate/2-thiouridine 02’Fluorocytosine triphosphate/5-bromouridine 1,818 2’Fluorocytosinetriphosphate/ 909 2’Fluorouridine triphosphate 2’Fluoroguaninetriphosphate/5-methylcytidine 0 2’Fluoroguanine triphosphate/ 05-methylcytidine/pseudouridine 2’Fluoroguanine triphosphate / 1,8185-methylcytidine/N1-methyl-pseudouridine 2’Fluoroguaninetriphosphate/pseudouridine 1,136 2’Fluoroguaninetriphosphate/2’Fluorocytosine 0 triphosphate/N1-methyl-pseudouridine5-methylcytidine/pseudouridine 617,7275-methylcytidine/N1-methyl-pseudouridine 747,0455-methylcytidine/pseudouridine 475,4555-methylcytidine/N1-methyl-pseudouridine 689,0915-methylcytosine/N1-methyl-pseudouridine, 848,409 Control 15-methylcytosine/N1-methyl-pseudouridine, 581,818 Control 2 Mock 682Untreated 0 Luciferase 2’Fluorocytosine triphosphate 0 Luciferase2’Fluorouridine triphosphate 0

Example 78 Screening of Chemistries

The tables listed in below (Tables 126-128) summarize much of the invitro and in vitro screening data with the different compounds presentedin the previous examples. A good correlation exists between cell-basedand cell-free translation assays. The same chemistry substitutionsgenerally show good concordance whether tested in the context ofluciferase or G-CSF mRNA. Lastly, N1-methyl-pseudouridine containingmRNA show a very high level of protein expression with little to nodetectable cytokine stimulation in vitro and in vivo, and is superior tomRNA containing pseudouridine both in vitro and in vivo.

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 33897; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) weremodified with naturally and non-naturally occurring chemistriesdescribed in Tables 126 and 127 or combination chemistries described inTable 128 and tested using methods described herein.

In Tables 126 and 127, “*” refers to in vitro transcription reactionusing a mutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No.DS010925) (Epicentre®, Madison, Wis.); “**” refers to the second resultin vitro transcription reaction using a mutant T7 polymerase(Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®,Madison, Wis.); “***” refers to production seen in cell freetranslations (rabbit reticulocyte lysates); the protein production ofHeLa is judged by “+,” “+/−” and “−”; when referring to G-CSF PBMC“++++” means greater than 6,000 pg/ml G-CSF, “+++” means greater than3,000 pg/ml G-CSF, “++” means greater than 1,500 pg/ml G-CSF, “+” meansgreater than 300 pg/ml G-CSF, “+/−” means 150-300 pg/ml G-CSF and thebackground was about 110 pg/ml; when referring to cytokine PBMC “++++”means greater than 1,000 pg/ml interferon-alpha (IFN-alpha), “+++” meansgreater than 600 pg/ml IFN-alpha, “++” means greater than 300 pg/mlIFN-alpha, “+” means greater than 100 pg/ml IFN-alpha, “−” means lessthan 100 pg/ml and the background was about 70 pg/ml; and “NT” means nottested. In Table 1277, the protein production was evaluated using amutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No.DS010925) (Epicentre®, Madison, Wis.).

TABLE 126 Naturally Occurring Protein Cytokines In In Protein Protein(G- (G- Vivo Vivo Common Name IVT IVT (Luc; (G-CSF; CSF; CSF; ProteinProtein (symbol) (Luc) (G-CSF) HeLa) HeLa) PBMC) PBMC) (Luc) (G-CSF)1-methyladenosine Fail Pass NT − +/− ++ NT NT (m¹A) N⁶- Pass Pass − −+/− ++++ NT NT methyladenosine (m⁶A) 2′-O- Fail* Not NT NT NT NT NT NTmethyladenosine Done (Am) 5-methylcytidine Pass Pass + + + ++ + NT (m⁵C)2′-O- Fail* Not NT NT NT NT NT NT methylcytidine Done (Cm)2-thiocytidine (s²C) Fail Fail NT NT NT NT NT NT N⁴-acetylcytidine PassPass + + +/− +++ + NT (ac⁴C) 5-formylcytidine Pass Pass −*** −*** − + NTNT (f⁵C) 2′-O- Fail* Not NT NT NT NT NT NT methylguanosine Done (Gm)inosine (I) Fail Fail NT NT NT NT NT NT pseudouridine (Y) Pass Pass + +++ + + NT 5-methyluridine Pass Pass + + +/− + NT NT (m⁵U)2′-O-methyluridine Fail* Not NT NT NT NT NT NT (Um) Done 1- Pass Pass +Not ++++ − + NT methylpseudouridine Done (m¹Y) 2-thiouridine (s²U) PassPass − + + + NT NT 4-thiouridine (s⁴U) Fail Pass + +/− ++ NT NT5-methoxyuridine Pass Pass + + ++ − + NT (mo⁵U) 3-methyluridine FailFail NT NT NT NT NT NT (m³U)

TABLE 127 Non-Naturally Occurring In Protein Protein Protein CytokinesIn Vivo Vivo Common IVT IVT (Luc; (G-CSF; (G-CSF; (G-CSF; ProteinProtein Name (Luc) (G-CSF) HeLa) HeLa) PBMC) PBMC) (Luc) (G-CSF)2′-F-ara- Fail Fail NT NT NT NT NT NT guanosine 2′-F-ara- Fail Fail NTNT NT NT NT NT adenosine 2′-F-ara- Fail Fail NT NT NT NT NT NT cytidine2′-F-ara- Fail Fail NT NT NT NT NT NT uridine 2′-F-guanosine Fail/ Pass/+** +/− − + + NT Pass* Fail** * 2′-F-adenosine Fail/ Fail/ −** NT NT NTNT NT Pass* Fail** * 2′-F-cytidine Fail/ Fail/ +** NT NT NT + NT Pass*Pass** * 2′-F-uridine Fail/ Pass/ +** + +/− + − NT Pass* Pass** *2′-OH-ara- Fail Fail NT NT NT NT NT NT guanosine 2′-OH-ara- Not Not NTNT NT NT NT NT adenosine Done Done 2′-OH-ara- Fail Fail NT NT NT NT NTNT cytidine 2′-OH-ara- Fail Fail NT NT NT NT NT NT uridine 5-Br-UridinePass Pass + + + + + 5-(2- Pass Pass − − +/− − carbomethoxy vinyl)Uridine 5-[3-(1-E- Pass Pass − + + − Propenylamino) Uridine (aka Chem 5)N6-(19- Fail Fail NT NT NT NT NT NT Amino- pentaoxanona decyl) A 2- FailFail NT NT NT NT NT NT Dimethylamino guanosine 6-Aza-cytidine Fail FailNT NT NT NT NT NT a-Thio- Pass Pass + + +/− +++ NT NT cytidine Pseudo-NT NT NT NT NT NT NT NT isocytidine 5-Iodo-uridine NT NT NT NT NT NT NTNT a-Thio-uridine NT NT NT NT NT NT NT NT 6-Aza-uridine NT NT NT NT NTNT NT NT Deoxy- NT NT NT NT NT NT NT NT thymidine a-Thio NT NT NT NT NTNT NT NT guanosine 8-Oxo- NT NT NT NT NT NT NT NT guanosine O6-Methyl-NT NT NT NT NT NT NT NT guanosine 7-Deaza- NT NT NT NT NT NT NT NTguanosine 6-Chloro- NT NT NT NT NT NT NT NT purine a-Thio- NT NT NT NTNT NT NT NT adenosine 7-Deaza- NT NT NT NT NT NT NT NT adenosine 5-iodo-NT NT NT NT NT NT NT NT cytidine

In Table 128, the protein production of HeLa is judged by “+,” “+/−” and“−”; when referring to G-CSF PBMC “++++” means greater than 6,000 pg/mlG-CSF, “+++” means greater than 3,000 pg/ml G-CSF, “++” means greaterthan 1,500 pg/ml G-CSF, “+” means greater than 300 pg/ml G-CSF, “+/−”means 150-300 pg/ml G-CSF and the background was about 110 pg/ml; whenreferring to cytokine PBMC “++++” means greater than 1,000 pg/mlinterferon-alpha (IFN-alpha), “+++” means greater than 600 pg/mlIFN-alpha, “++” means greater than 300 pg/ml IFN-alpha, “+” meansgreater than 100 pg/ml IFN-alpha, “−” means less than 100 pg/ml and thebackground was about 70 pg/ml; “WT” refers to the wild type T7polymerase, “MT” refers to mutant T7 polymerase (Durascribe® T7Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.) and“NT” means not tested.

TABLE 128 Combination Chemistry Protein Protein In IVT Protein (G- (G-Cytokines Vivo Cytidine Uridine IVT (G- (Luc; CSF; CSF; (G-CSF; Proteinanalog analog Purine Luc CSF) HeLa) HeLa) PBMC) PBMC) (Luc) N4-pseudouridine A, G Pass Pass + + NT NT + acetylcytidine WT WT N4- N1- A,G Pass Pass + + NT NT + acetylcytidine methyl- WT WT pseudouridine 5- 5-A, G Pass Pass + + NT NT + methylcytidine methoxyuridine WT WT 5- 5- A,G Pass Pass Not + NT NT methylcytidine bromouridine WT WT Done 5- N1- A,G Pass Pass + + NT NT + methylcytidine methyluridine WT WT 5- 50% 2- A,G Pass Pass + NT NT NT + methylcytidine thiuridine; WT WT 50% uridine 5-100% 2- A, G Pass Pass − + NT NT methylcytidine thiouridine WT WT 5-pseudouridine A, G Pass Pass + + ++ + + methylcytidine WT WT 5- N1- A, GPass Pass + + ++++ − + methylcytidine methyl- WT WT pseudouridine N4- 2-A, G Not Pass Not + NT NT NT acetylcytidne thiouridine Done WT Done N4-5- A, G Not Pass Not + NT NT NT acetylcytidine bromouridine Done WT DoneN4- 2 A, G Pass Pass − + NT NT NT acetylcytidine Fluorouridinetriphosphate 5- 2 A, G Pass Pass − + NT NT NT methylcytidineFluorouridine triphosphate 2 pseudouridine A, G Pass Pass − + NT NT NTFluorocytosine triphosphate 2 N1- A, G Pass Pass − +/− NT NT NTFluorocytosine methyl- triphosphate pseudouridine 2 2- A, G Pass Pass −− NT NT NT Fluorocytosine thiouridine triphosphate 2 5- A, G Pass Pass −+/− NT NT NT Fluorocytosine bromouridine triphosphate 2 2 A, G Pass Pass− +/− NT NT NT Fluorocytosine Fluorouridine triphosphate triphosphate 5-uridine A, 2 Pass Pass − − NT NT NT methylcytidine Fluoro GTP 5-pseudouridine A, 2 Pass Pass − − NT NT NT methylcytidine Fluoro GTP 5-N1- A, 2 Pass Pass − +/− NT NT NT methylcytidine methyl- Fluoropseudouridine GTP 2 pseudouridine A, 2 Pass Pass − +/− NT NT NTFluorocytosine Fluoro triphosphate GTP 2 N1- A, 2 Pass Pass − − NT NT NTFluorocytosine methyl- Fluoro triphosphate pseudouridine GTP

Example 79 2′Fluoro Chemistries in PBMC

The ability of G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO:33897; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1) to trigger innate an immune response wasdetermined by measuring interferon-alpha (IFN-alpha) and tumor necrosisfactor-alpha (TNF-alpha) production. Use of in vitro PBMC cultures is anaccepted way to measure the immunostimulatory potential ofoligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102) andtransfection methods are described herein. Shown in Table 129 are theaverage from 2 or 3 separate PBMC donors of the interferon-alpha(IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) production overtime as measured by specific ELISA. Controls of R848, P(I)P(C), LPS andLipofectamine 2000 (L2000) were also analyzed.

With regards to innate immune recognition, while both modified mRNAchemistries largely prevented IFN-alpha and TNF-alpha productionrelative to positive controls (R848, P(I)P(C)), 2′fluoro compoundsreduce IFN-alpha and TNF-alpha production even lower than othercombinations and N4-acetylcytidine combinations raised the cytokineprofile.

TABLE 129 IFN-alpha and TNF-alpha IFN-alpha: TNF-alpha: 3 Donor 2 DonorAverage Average (pg/ml) (pg/ml) L2000 1 361 P(I)P(C) 482 544 R848 458,235 LPS 0 6,889 N4-acetylcytidine/pseudouridine 694 528N4-acetylcytidine/N1-methyl-pseudouridine 307 2835-methylcytidine/5-methoxyuridine 0 411 5-methylcytidine/5-bromouridine0 270 5-methylcytidine/5-methyluridine 456 4285-methylcytidine/2-thiouridine 274 277 N4-acetylcytidine/2-thiouridine 0285 N4-acetylcytidine/5-bromouridine 44 4035-methylcytidine/pseudouridine 73 3325-methylcytidine/N1-methyl-pseudouridine 31 280N4-acetylcytidine/2′fluorouridine triphosphate 35 325-methylcytodine/2′fluorouridine triphosphate 24 0 2′fluorocytidinetriphosphate/N1-methyl- 0 11 pseudouridine 2′fluorocytidinetriphosphate/2-thiouridine 0 02′fluorocytidine/triphosphate5-bromouridine 12 2 2′fluorocytidinetriphosphate/2′fluorouridine 11 0 triphosphate 2′fluorocytidinetriphosphate/5-methylcytidine 14 23 2′fluorocytidine triphosphate/5- 621 methylcytidine/pseudouridine 2′fluorocytidine triphosphate/5- 3 15methylcytidine/N1-methyl-pseudouridine 2′fluorocytidinetriphosphate/pseudouridine 0 4 2′fluorocytidine triphosphate/N1-methyl-6 20 pseudouridine 5-methylcytidine/pseudouridine 82 185-methylcytidine/N1-methyl-pseudouridine 35 3

Example 80 Modified mRNA with a Tobacco Etch Virus 5′UTR

A 5′ untranslated region (UTR) may be provided as a flanking region.Multiple 5′ UTRs may be included in the flanking region and may be thesame or of different sequences. Any portion of the flanking regions,including none, may be codon optimized and any may independently containone or more different structural or chemical modifications, beforeand/or after codon optimization.

The 5′ UTR may comprise the 5′UTR from the tobacco etch virus (TEV).Variants of 5′ UTRs may be utilized wherein one or more nucleotides areadded or removed to the termini, including A, T, C or G.

Example 81 Expression of PLGA Formulated mRNA

A. Synthesis and Characterization of Luciferase PLGA Microspheres

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fullymodified with 5-methylcytosine and N1-methyl pseudouridine, modifiedwith 25% of uridine replaced with 2-thiouridine and 25% of cytosinereplaced with 5-methylcytosine, fully modified with N1-methylpseudouridine, or fully modified with pseudouridine was reconstituted in1× TE buffer and then formulated in PLGA microspheres. PLGA microsphereswere synthesized using the water/oil/water double emulsification methodsknown in the art using PLGA-ester cap (Lactel, Cat #B6010-2, inherentviscosity 0.55-0.75, 50:50 LA:GA), polyvinylalcohol (PVA) (Sigma, Cat#348406-25G, MW 13-23 k) dichloromethane and water. Briefly, 0.4 ml ofmRNA in TE buffer at 4 mg/ml (W1) was added to 2 ml of PLGA dissolved indichloromethane (DCM) (O1) at a concentration of 200 mg/ml of PLGA. TheW1/O1 emulsion was homogenized (IKA Ultra-Turrax Homogenizer, T18) for30 seconds at speed 5 (19,000 rpm). The W1/O1 emulsion was then added to250 ml 1% PVA (W2) and homogenized for 1 minute at speed 5 (˜19,000rpm). Formulations were left to stir for 3 hours, then passed through a100 μm nylon mesh strainer (Fisherbrand Cell Strainer, Cat #22-363-549)to remove larger aggregates, and finally washed by centrifugation (10min, 9,250 rpm, 4° C.). The supernatant was discarded and the PLGApellets were resuspended in 5-10 ml of water, which was repeated 2×.After washing and resuspension with water, 100-200 μl of a PLGAmicrospheres sample was used to measure particle size of theformulations by laser diffraction (Malvern Mastersizer2000). The washedformulations were frozen in liquid nitrogen and then lyophilized for 2-3days.

After lyophilization, ˜10 mg of PLGA MS were weighed out in 2 mleppendorf tubes and deformulated by adding 1 ml of DCM and letting thesamples shake for 2-6 hrs. The mRNA was extracted from the deformulatedPLGA microspheres by adding 0.5 ml of water and shaking the sampleovernight. Unformulated luciferase mRNA in TE buffer (unformulatedcontrol) was spiked into DCM and went through the deformulation process(deformulation control) to be used as controls in the transfectionassay. The encapsulation efficiency, weight percent loading and particlesize are shown in Table 130. Encapsulation efficiency was calculated asmg of mRNA from deformulation of PLGA microspheres divided by theinitial amount of mRNA added to the formulation. Weight percent loadingin the formulation was calculated as mg of mRNA from deformulation ofPLGA microspheres divided by the initial amount of PLGA added to theformulation.

TABLE 130 PLGA Characteristics Parti- Theoretical Actual cle mRNA mRNASize Chemical Encapsulation Loading Loading (D50, Modifications SampleID Efficiency (%) (wt %) (wt %) um) Fully modified 43-66A 45.8 0.4 0.1833.4 with 5- 43-66B 29.6 0.12 27.7 methylcytosine 43-66C 25.5 0.10 27.1and N1-methyl pseudouridine 25% of uridine 43-67A 34.6 0.4 0.14 29.9replaced with 2- 43-67B 22.8 0.09 30.2 thiouridine and 43-67C 23.9 0.1025.1 25% of cytosine replaced with 5- methylcytosine Fully modified43-69A 55.8 0.4 0.22 40.5 with N1-methyl 43-69B 31.2 0.12 41.1pseudouridine 43-69C 24.9 0.10 46.1 Fully modified 43-68-1 49.3 0.4 0.2034.8 with 43-68-2 37.4 0.15 35.9 pseudouridine 43-68-3 45.0 0.18 36.5

B. Protein Expression of Modified mRNA Encapsulated in PLGA Microspheres

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 83 ng ofthe deformulated luciferase mRNA PLGA microsphere samples, deformulatedluciferase mRNA control (Deform control), or unformulated luciferasemRNA control (Unfomul control) was diluted in a 10 ul final volume ofOPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as a transfectionreagent and 0.2 ul was diluted in a 10 ul final volume of OPTI-MEM.After 5 min of incubation at room temperature, both solutions werecombined and incubated an additional 15 min at room temperature. Then 20ul of the combined solution was added to 100 ul of cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After an 18 to 22 hour incubation, cells expressing luciferase werelysed with 100 ul Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The background signal of the plateswithout reagent was about 200 relative light units per well. The platereader was a BioTek Synergy H1 (BioTek, Winooski, Vt.).

Cells were harvested and the bioluminescence (in relative light units,RLU) for each sample is shown in Table 131. Transfection of thesesamples confirmed that the varied chemistries of luciferase mRNA isstill able to express luciferase protein after PLGA microsphereformulation.

TABLE 131 Chemical Modifications Chemical Biolum. Modifications SampleID (RLU) Fully modified with Deform contol 164266.5 5-methylcytosineUnformul control 113714 and N1-methyl- 43-66A 25174 pseudouridine 43-66B25359 43-66C 20060 25% of uridine Deform contol 90816.5 replaced with 2-Unformul control 129806 thiouridine and 25% 43-67A 38329.5 of cytosinereplaced 43-67B 8471.5 with 5- 43-67C 10991.5 methylcytosine Fullymodified with Deform contol 928093.5 N1-methyl- Unformul control1512273.5 pseudouridine 43-69A 1240299.5 43-69B 748667.5 43-69C 1193314Fully modified with Deform contol 154168 pseudouridine Unformul control151581 43-68-1 120974.5 43-68-2 107669 43-68-3 97226

Example 82 In Vitro Studies of Factor IX

A. Serum-Free Media

Human Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 33901; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) wastransfected in serum-free media. The cell culture supernatant wascollected and subjected to trypsin digestion before undergoing2-dimensional HPLC separation of the peptides. Matrix-assisted laserdesorption/ionization was used to detect the peptides. 8 peptides weredetected and 7 of the detected peptides are unique to Factor IX. Theseresults indicate that the mRNA transfected in the serum-free media wasable to express full-length Factor IX protein.

B. Human Embryonic Kidney (HEK) 293A Cells

250 ng of codon optimized Human Factor IX mRNA (mRNA sequence shown inSEQ ID NO: 33901; fully modified with 5-methylcytosine andpseudouridine; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1) was transfected into HEK 293A cells (150,000cells/well) using Lipofectamine 2000 in DMEM in presence of 10% FBS. Thetransfection complexes were removed 3 hours after transfection. Cellswere harvested at 3, 6, 9, 12, 24, 48 and 72 hours after transfection.Total RNA was isolated and used for cDNA synthesis. The cDNA wassubjected to analysis by quantitative Real-Time PCR using codonoptimized Factor IX specific primer set. Human hypoxanthinephosphoribosyltransferase 1 (HPRT) level was used for normalization. Thedata is plotted as a percent of detectable mRNA considering the mRNAlevel as 100% at the 3 hour time point. The half-life of Factor IXmodified mRNA fully modified with 5-methylcytosine and pseudouridine inhuman embryonic kidney 293 (HEK293) cells is about 8-10 hours.

Example 83 Saline Formulation: Subcutaneous Administration

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 33897;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) andhuman EPO modified mRNA (mRNA sequence shown in SEQ ID NO: 33900; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and pseudouridine), wereformulated in saline and delivered to mice via intramuscular (IM)injection at a dose of 100 ug.

Controls included Luciferase (mRNA sequence shown in SEQ ID NO: 33907;polyA tail of approximately 160 nucleotides not shown in sequence;5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine)) orthe formulation buffer (F.Buffer). The mice were bled at 13 hours afterthe injection to determine the concentration of the human polypeptide inserum in pg/mL. (G-CSF groups measured human G-CSF in mouse serum andEPO groups measured human EPO in mouse serum). The data are shown inTable 132.

mRNA degrades rapidly in serum in the absence of formulation suggestingthe best method to deliver mRNA to last longer in the system is byformulating the mRNA. As shown in Table 132, mRNA can be deliveredsubcutaneously using only a buffer formulation.

TABLE 132 Dosing Regimen Average Protein Dose Product Vol. Dosing pg/mL,Group Treatment (μl/mouse) Vehicle serum G-CSF G-CSF 100 F. buffer 45G-CSF Luciferase 100 F. buffer 0 G-CSF F. buffer 100 F. buffer 2.2 EPOEPO 100 F. buffer 72.03 EPO Luciferase 100 F. buffer 26.7 EPO F. buffer100 F. buffer 13.05

Example 84 Intravitreal Delivery

mCherry modified mRNA (mRNA sequence shown in SEQ ID NO: 33898; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) andluciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) formulatedin saline was delivered intravitreally in rats as described in Table133. The sample was compared against a control of saline only deliveredintravitreally.

TABLE 133 Dosing Chart Dose Level Dose Treatment (μg modified volumeRight Eye Left Eye Group No. RNA/eye) (μL/eye) (OD) (OS) Control 0 5Delivery Delivery buffer only buffer only Modified RNA in 10 5 mCherryLuciferase delivery buffer

The formulation will be administered to the left or right eye of eachanimal on day 1 while the animal is anesthetized. On the day prior toadministration gentamicin ophthalmic ointment or solution was applied toboth eyes twice. The gentamicin ophthalmic ointment or solution was alsoapplied immediately following the injection and on the day following theinjection. Prior to dosing, mydriatic drops (1% tropicamide and/or 2.5%phenylephrine) are applied to each eye.

18 hours post dosing the eyes receiving the dose of mCherry and deliverybuffer are enucleated and each eye was separately placed in a tubecontaining 10 mL 4% paraformaldehyde at room temperature for overnighttissue fixation. The following day, eyes will be separately transferredto tubes containing 10 mL of 30% sucrose and stored at 21° C. until theywere processed and sectioned. The slides prepared from differentsections were evaluated under F-microscopy. Positive expression was seenin the slides prepared with the eyes administered mCherry modified mRNAand the control showed no expression.

Example 85 In Vivo Cytokine Expression Study

Mice were injected intramuscularly with 200 ug of G-CSF modified mRNA(mRNA sequence shown in SEQ ID NO: 33897; polyA tail of approximately160 nucleotides not shown in sequence) which was unmodified with a5′cap, Cap1 (unmodified), fully modified with 5-methylcytosine andpseudouridine and a 5′cap, Cap1 (Gen1) or fully modified with5-methylcytosine and N1-methylpseudouridine and a 5′cap, Cap1 (Gen2 cap)or no cap (Gen2 uncapped). Controls of R-848, 5% sucrose and untreatedmice were also analyzed. After 8 hours serum was collected from the miceand analyzed for interferon-alpha (IFN-alpha) expression. The resultsare shown in Table 134.

TABLE 134 IFN-alpha Expression Formulation IFN-alpha (pg/ml) G-CSFunmodified 67.012 G-CSF Gen1 8.867 G-CSF Gen2 cap 0 G-CSF Gen2 uncapped0 R-848 40.971 5% sucrose 1.493 Untreated 0

Example 86 In Vitro Expression of VEGF Modified mRNA

HEK293 cells were transfected with modified mRNA (mmRNA) VEGF-A (mRNAsequence shown in SEQ ID NO: 33910; polyA tail of approximately 160nucleotides not shown in sequence; 5′cap, Cap1; fully modified with5-methylcytosine and pseudouridine) which had been complexed withLipofectamine 2000 from Invitrogen (Carlsbad, Calif.) at theconcentration shown in Table 135. The protein expression was detected byELISA and the protein (pg/ml) is shown in Table 135.

TABLE 135 Protein Expression Amount 10 ng 2.5 ng 625 pg 156 pg 39 pg 10pg 2 pg 610 fg Trans- fected Protein 10495 10038 2321.23 189.6 0 0 0 0(pg/ml)

Example 87 In Vitro Screening in HeLa Cells of GFP

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 37.5 ng or 75ng of Green Fluorescent protein (GFP) modified RNA (mRNA sequence shownin SEQ ID NO: 33909; polyA tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap1) with the chemical modificationdescribed in Table 136, were diluted in 10 ul final volume of OPTI-MEM(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul were diluted in 10 ul final volume of OPTI-MEM. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minute at room temperature. Then the 20ul combined solution was added to the 100 ul cell culture mediumcontaining the HeLa cells and incubated at room temperature.

After an 18 to 22 hour incubation cells expressing luciferase were lysedwith 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.) accordingto manufacturer instructions. Aliquots of the lysates were transferredto white opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100 ul complete luciferase assay solution (Promega,Madison, Wis.). The median fluorescence intensity (MFI) was determinedfor each chemistry and is shown in Table 136.

These results demonstrate that GFP fully modified withN1-methylpseudouridine and 5-methylcytosine produces more protein inHeLa cells compared to the other chemistry. Additionally the higher doseof GFP administered to the cells resulted in the highest MFI value.

TABLE 136 Mean Fluorescence Intensity 37.5 ng 75 ng Chemistry MFI MFI Nomodifications 97400 89500 5-methylcytosine/pseudouridine 324000 7150005-methylcytosine/N1-methyl-pseudouridine 643000 1990000

Example 88 Homogenization

Different luciferase mRNA solutions (as described in Table 137 where “X”refers to the solution containing that component) (mRNA sequence shownin SEQ ID NO: 33907; polyA tail of approximately 160 nucleotides notshown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) were evaluated to test the percent yield of the differentsolutions, the integrity of the mRNA by bioanalyzer, and the proteinexpression of the mRNA by in vitro transfection. The mRNA solutions wereprepared in water, 1× TE buffer at 4 mg/ml as indicated in Table 137,and added to either dichloromethane (DCM) or DCM containing 200 mg/ml ofpoly(lactic-co-glycolic acid) (PLGA) (Lactel, Cat #B6010-2, inherentviscosity 0.55-0.75, 50:50 LA:GA) to achieve a final mRNA concentrationof 0.8 mg/ml. The solutions requiring homogenization were homogenizedfor 30 seconds at speed 5 (approximately 19,000 rpm) (IKA Ultra-TurraxHomogenizer, T18). The mRNA samples in water, dichloromethane andpoly(lactic-co-glycolic acid) (PLGA) were not recoverable (NR). Allsamples, except the NR samples, maintained integrity of the mRNA asdetermined by bioanalyzer (Bio-rad Experion).

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 250 ngof luciferase mRNA from the recoverable samples was diluted in a 10 ulfinal volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as atransfection reagent and 0.2 ul was diluted in a 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 20 ul of the combined solution was added to 100 ul ofcell culture medium containing the HeLa cells. The plates were thenincubated as described before. Controls luciferase mRNA (luciferase mRNAformulated in saline) (Control) and untreated cells (Untreat.) were alsoevaluated. Cells were harvested and the bioluminescence average (inphotons/second) (biolum. (p/s)) for each signal is also shown in Table137. The recoverable samples all showed activity of luciferase mRNA whenanalyzed.

After an 18 to 22 hour incubation, cells expressing luciferase werelysed with 100 ul Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The background signal of the plateswithout reagent was about 200 relative light units per well. The platereader was a BioTek Synergy H1 (BioTek, Winooski, Vt.).

Cells were harvested and the bioluminescence average (in relative lightunits, RLU) (biolum. (RLU)) for each signal is also shown in Table 137.The recoverable samples all showed activity of luciferase mRNA whenanalyzed.

TABLE 137 Solutions Solution 1x TE DCM/ Yield Biolum. No. Water BufferDCM PLGA Homogenizer (%) (RLU) 1 X 96 5423780 2 X X 95 4911950 3 X X 922367230 4 X X 90 4349410 5 X X X 66 4145340 6 X X X 71 3834440 7 X X XNR n/a 8 X X X 24 3182080 9 X X NR n/a 10 X X 79 3276800 11 X X 795563550 12 X X 79 4919100 Control 2158060 Untreat. 3530

Example 89 TE Buffer and Water Evaluation

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) was reconstituted inwater or TE buffer as outlined in Table 138 and then formulated in PLGAmicrospheres. PLGA microspheres were synthesized using thewater/oil/water double emulsification methods known in the art usingPLGA (Lactel, Cat #B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA),polyvinylalcohol (PVA) (Sigma, Cat #348406-25G, MW 13-23 k)dichloromethane and water. Briefly, 0.2 to 0.6 ml of mRNA in water or TEbuffer at a concentration of 2 to 6 mg/ml (W1) was added to 2 ml of PLGAdissolved in dichloromethane (DCM) (O1) at a concentration of 100 mg/mlof PLGA. The W1/O1 emulsion was homogenized (IKA Ultra-TurraxHomogenizer, T18) for 30 seconds at speed 5 (˜19,000 rpm). The W1/O1emulsion was then added to 250 ml 1% PVA (W2) and homogenized for 1minute at speed 5 (˜19,000 rpm). Formulations were left to stir for 3hours, then passed through a 100 μm nylon mesh strainer (FisherbrandCell Strainer, Cat #22-363-549) to remove larger aggregates, and finallywashed by centrifugation (10 min, 9,250 rpm, 4° C.). The supernatant wasdiscarded and the PLGA pellets were resuspended in 5-10 ml of water,which was repeated 2×. The washed formulations were frozen in liquidnitrogen and then lyophilized for 2-3 days. After lyophilization, ˜10 mgof PLGA MS were weighed out in 2 ml eppendorf tubes and deformulated byadding 1 ml of DCM and letting the samples shake for 2-6 hrs. mRNA wasextracted from the deformulated PLGA microspheres by adding 0.5 ml ofwater and shaking the sample overnight. Unformulated luciferase mRNA inwater or TE buffer (deformulation controls) was spiked into DCM and wentthrough the deformulation process to be used as controls in thetransfection assay.

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 100 ngof the deformulated luciferase mRNA samples was diluted in a 10 ul finalvolume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine2000 (LifeTechnologies, Grand Island, N.Y.) was used as a transfectionreagent and 0.2 ul was diluted in a 10 ul final volume of OPTI-MEM.After 5 minutes of incubation at room temperature, both solutions werecombined and incubated an additional 15 minutes at room temperature.Then 20 ul of the combined solution was added to 100 ul of cell culturemedium containing the HeLa cells. The plates were then incubated asdescribed before.

After 18 to 22 hour incubation, cells expressing luciferase were lysedwith 100 ul Passive Lysis Buffer (Promega, Madison, Wis.) according tomanufacturer instructions. Aliquots of the lysates were transferred towhite opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100 ul complete luciferase assay solution (Promega,Madison, Wis.). The background signal of the plates without reagent wasabout 200 relative light units per well. The plate reader was a BioTekSynergy H1 (BioTek, Winooski, Vt.). To determine the activity of theluciferase mRNA from each formulation, the relative light units (RLU)for each formulation was divided by the RLU of the appropriate mRNAdeformulation control (mRNA in water or TE buffer). Table 138 shows theactivity of the luciferase mRNA. The activity of the luciferase mRNA inthe PLGA microsphere formulations (Form.) was substantially improved byformulating in TE buffer versus water.

TABLE 138 Formulations Activ- Theo- ity mRNA W1 retical Actual (% ofconc. Solvent Total mRNA mRNA W1 deform. (mg/ volume mRNA LoadingLoading Sol- con- Form. ml) (ul) (ug) (wt %) (wt %) vent trol) PLGA A 4400 1600 0.80 0.14 Water 12.5% PLGA B 4 200 800 0.40 0.13 Water  1.3%PLGA C 4 600 2400 1.20 0.13 Water 12.1% PLGA D 2 400 800 0.40 0.07 Water 1.3% PLGA E 6 400 2400 1.20 0.18 TE 38.9% Buffer PLGA F 4 400 1600 0.800.16 TE 39.7% Buffer PLGA G 4 400 1600 0.80 0.10 TE 26.6% Buffer

Example 90 Chemical Modifications on mRNA

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(Life Technologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 83 ng ofLuciferase modified RNA (mRNA sequence shown SEQ ID NO: 33907; polyAtail of approximately 140 nucleotides not shown in sequence; 5′cap,Cap1) with the chemical modification described in Table 139, werediluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul were diluted in 10 ul finalvolume of OPTI-MEM. After 5 minutes of incubation at room temperature,both solutions were combined and incubated an additional 15 minute atroom temperature. Then the 20 ul combined solution was added to the 100ul cell culture medium containing the HeLa cells and incubated at roomtemperature.

After 18 to 22 hours of incubation cells expressing luciferase werelysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The lysate volumes were adjusted ordiluted until no more than 2 mio relative light units (RLU) per wellwere detected for the strongest signal producing samples and the RLUsfor each chemistry tested are shown in Table 139. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of theplates without reagent was about 200 relative light units per well.

TABLE 139 Chemical Modifications Sample RLU Untreated 336 UnmodifiedLuciferase 33980 5-methylcytosine and pseudouridine 16012345-methylcytosine and N1-methyl-pseudouridine 421189 25% cytosinesreplaced with 5-methylcytosine and 25% of uridines 222114 replaced with2-thiouridine N1-methyl-pseudouridine 3068261 Pseudouridine 140234N4-Acetylcytidine 1073251 5-methoxyuridine 219657 5-Bromouridine 6787N4-Acetylcytidineand N1-methyl-pseudouridine 976219 5-methylcytosine and5-methoxyuridine 66621 5-methylcytosine and 2′fluorouridine 11333

Example 91 Intramuscular and Subcutaneous Administration of ModifiedmRNA

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 140 nucleotides not shown in sequence; 5′cap,Cap1) fully modified with 5-methylcytosine and pseudouridine (5mC/pU),fully modified with 5-methylcytosine and N1-methyl-pseudouridine(5mC/N1mpU), fully modified with pseudouridine (pU), fully modified withN1-methyl-pseudouridine (N1mpU) or modified where 25% of the cytosinesreplaced with 5-methylcytosine and 25% of the uridines replaced with2-thiouridine (5mC/s2U) formulated in PBS (pH 7.4) was administered toBalb-C mice intramuscularly or subcutaneously at a dose of 2.5 mg/kg.The mice were imaged at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours,96 hours, 120 hours and 144 hours for intramuscular delivery and 2hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours and 120 hours forsubcutaneous delivery. Twenty minutes prior to imaging, mice wereinjected intraperitoneally with a D-luciferin solution at 150 mg/kg.Animals were then anesthetized and images were acquired with an IVISLumina II imaging system (Perkin Elmer). Bioluminescence was measured astotal flux (photons/second) of the entire mouse. The average total flux(photons/second) for intramuscular administration is shown in Table 140and the average total flux (photons/second) for subcutaneousadministration is shown in Table 141. The background signal was 3.79E+05(p/s). The peak expression for intramuscular administration was seenbetween 24 and 48 hours for all chemistry and expression was stilldetected at 144 hours. For subcutaneous delivery the peak expression wasseen at 2-8 hours and expression was detected at 72 hours.

TABLE 140 Intramuscular Administration 5 mC/pU 5 mC/N1mpU 5 mC/s2U pUN1mpU Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s)  2 hours1.98E+07 4.65E+06 4.68E+06 2.33E+06 3.66E+07  8 hours 1.42E+07 3.64E+063.78E+06 8.07E+06 7.21E+07 24 hours 2.92E+07 1.22E+07 3.35E+07 1.01E+071.75E+08 48 hours 2.64E+07 1.01E+07 5.06E+07 7.46E+06 3.42E+08 72 hours2.18E+07 8.59E+06 3.42E+07 4.08E+06 5.83E+07 96 hours 2.75E+07 2.70E+062.38E+07 4.35E+06 7.15E+07 120 hours  2.19E+07 1.60E+06 1.54E+071.25E+06 3.87E+07 144 hours  9.17E+06 2.19E+06 1.14E+07 1.86E+065.04E+07

TABLE 141 Subcutaneous Administration 5 mC/pU 5 mC/N1mpU 5 mC/s2U pUN1mpU Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s)  2 hours5.26E+06 4.54E+06 9.34E+06 2.43E+06 2.80E+07  8 hours 2.32E+06 8.75E+058.15E+06 2.12E+06 3.09E+07 24 hours 2.67E+06 5.49E+06 3.80E+06 2.24E+061.48E+07 48 hours 1.22E+06 1.77E+06 3.07E+06 1.58E+06 1.24E+07 72 hours1.12E+06 8.00E+05 8.53E+05 4.80E+05 2.29E+06 96 hours 5.16E+05 5.33E+054.30E+05 4.30E+05 6.62E+05 120 hours  3.80E+05 4.09E+05 3.21E+056.82E+05 5.05E+05

Example 92 Osmotic Pump Study

Prior to implantation, an osmotic pump (ALZET® Osmotic Pump 2001D,DURECT Corp. Cupertino, Calif.) is loaded with the 0.2 ml of 1×PBS (pH7.4) (PBS loaded pump) or 0.2 ml of luciferase modified mRNA (mRNAsequence shown in SEQ ID NO: 33907; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and N1-methylpseudouridine) at 1 mg/ml in 1×PBS (pH7.4) (Luciferase loaded pump) and incubated overnight in 1×PBS (pH 7.4)at 37° C.

Balb-C mice (n=3) are implanted subcutaneously with either the PBSloaded pump or the luciferase loaded pump and imaged at 2 hours, 8 hoursand 24 hours. As a control a PBS loaded pump is implanted subcutaneouslyand the mice are injected subcutaneously with luciferase modified mRNAin 1×PBS (PBS loaded pump; SC Luciferase) or an osmotic pump is notimplanted and the mice are injected subcutaneously with luciferasemodified mRNA in 1×PBS (SC Luciferase). The luciferase formulations areoutlined in Table 142.

TABLE 142 Luciferase Formulations Conc Inj. Vol. Amt Dose Group Vehicle(mg/ml) (ul) (ug) (mg/kg) PBS loaded pump; SC PBS 1.00 50 50 2.5Luciferase Luciferase loaded pump PBS 1.00 — 200 10.0 PBS loaded pumpPBS — — — — SC Luciferase PBS 1.00 50 50 2.5

Example 93 External Osmotic Pump Study

An external osmotic pump (ALZET® Osmotic Pump 2001D, DURECT Corp.Cupertino, Calif.) is loaded with the 0.2 ml of 1×PBS (pH 7.4) (PBSloaded pump) or 0.2 ml of luciferase modified mRNA (mRNA sequence shownin SEQ ID NO: 33907; polyA tail of approximately 140 nucleotides notshown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine andN1-methylpseudouridine) at 1 mg/ml in 1×PBS (pH 7.4) (luciferase loadedpump) and incubated overnight in 1×PBS (pH 7.4) at 37° C.

Using a catheter connected to the external PBS loaded pump or theluciferase loaded pump Balb-C mice (n=3) are administered theformulation. The mice are imaged at 2 hours, 8 hours and 24 hours. As acontrol an external PBS loaded pump is used and the mice are injectedsubcutaneously with luciferase modified mRNA in 1×PBS (PBS loaded pump;SC Luciferase) or the external pump is not used and the mice are onlyinjected subcutaneously with luciferase modified mRNA in 1×PBS (SCLuciferase). Twenty minutes prior to imaging, mice are injectedintraperitoneally with a D-luciferin solution at 150 mg/kg. Animals arethen anesthetized and images are acquired with an IVIS Lumina II imagingsystem (Perkin Elmer). Bioluminescence is measured as total flux(photons/second) of the entire mouse. The luciferase formulations areoutlined in Table 143 and the average total flux (photons/second).

TABLE 143 Luciferase Formulations Conc Inj. Vol. Amt Dose Group Vehicle(mg/ml) (ul) (ug) (mg/kg) PBS loaded pump; SC PBS 1.00 50 50 2.5Luciferase Luciferase loaded pump PBS 1.00 — 200 10.0 PBS loaded pumpPBS — — — — SC Luciferase PBS 1.00 50 50 2.5

Example 94 Fibrin Sealant Study

Fibrin sealant, such as Tisseel (Baxter Healthcare Corp., Deerfield,Ill.), is composed of fibrinogen and thrombin in a dual-barreledsyringe. Upon mixing, fibrinogen is converted to fibrin to form a fibrinclot in about 10 to 30 seconds. This clot can mimic the natural clottingmechanism of the body. Additionally a fibrin hydrogel is a threedimensional structure that can potentially be used in sustained releasedelivery. Currently, fibrin sealant is approved for application inhemostasis and sealing to replace conventional surgical techniques suchas suture, ligature and cautery.

The thrombin and fibrinogen components were loaded separately into adual barreled syringe. Balb-C mice (n=3) were injected subcutaneouslywith 50 ul of fibrinogen, 50 ul of thrombin and they were also injectedat the same site with modified luciferase mRNA (mRNA sequence shown inSEQ ID NO: 33907; polyA tail of approximately 140 nucleotides not shownin sequence; 5′cap, Cap1; fully modified with 5-methylcytosine andN1-methylpseudouridine) (Tisseel+Luciferase), 50 ul of fibrinogen and 50ul thrombin (Tisseel) or modified luciferase mRNA (Luciferase). Theinjection of fibrinogen and thrombin was done simultaneously using thedual-barreled syringe. The SC injection of luciferase was done 15minutes after the fibrinogen/thrombin injection to allow the fibrinhydrogel to polymerize (Tisseel+Luciferase group). A control group ofuntreated mice were also evaluated. The mice were imaged at 5 hours and24 hours. Twenty minutes prior to imaging, mice were injectedintraperitoneally with a D-luciferin solution at 150 mg/kg. Animals werethen anesthetized and images were acquired with an IVIS Lumina IIimaging system (Perkin Elmer). Bioluminescence was measured as totalflux (photons/second) of the entire mouse. The luciferase formulationsare outlined in Table 144 and the average total flux (photons/second) isshown in Table 145. The fibrin sealant was found to not interfere withimaging and the injection of luciferase and Tisseel showed expression ofluciferase.

TABLE 144 Luciferase Formulations Conc Inj. Vol. Amt Dose Group Vehicle(mg/ml) (ul) (ug) (mg/kg) Tisseel + Luciferase PBS 1.00 50 50 2.5Tisseel — — — — — Luciferase PBS 1.00 50 50 2.5 Untreated — — — — —

TABLE 145 Total Flux 5 Hours 24 Hours Group Flux (p/s) Flux (p/s)Tisseel + Luciferase 4.59E+05 3.39E+05 Tisseel 1.99E+06 1.06E+06Luciferase 9.94E+05 7.44E+05 Untreated 3.90E+05 3.79E+05

Example 95 Fibrin Containing mRNA Sealant Study

A. Modified mRNA and Calcium Chloride

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 33907; polyA tail of approximately 140 nucleotides not shown insequence; 5′cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with N1-methylpseudouridine isadded to calcium chloride. The calcium chloride is then used toreconstitute thrombin. Fibrinogen is reconstituted with fibrinolysisinhibitor solution per the manufacturer's instructions. Thereconstituted thrombin containing modified mRNA and fibrinogen is loadedinto a dual barreled syringe. Mice are injected subcutaneously with 50ul of fibrinogen and 50 ul of thrombin containing modified mRNA or theywere injected with 50 ul of PBS containing an equivalent dose ofmodified luciferase mRNA. A control group of untreated mice is alsoevaluated. The mice are imaged at predetermined intervals to determinethe average total flux (photons/second).

B. Lipid Nanoparticle Formulated Modified mRNA and Calcium Chloride

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 33907; polyA tail of approximately 140 nucleotides not shown insequence; 5′cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with N1-methylpseudouridine isformulated in a lipid nanoparticle is added to calcium chloride. Thecalcium chloride is then used to reconstitute thrombin. Fibrinogen isreconstituted with fibrinolysis inhibitor solution per themanufacturer's instructions. The reconstituted thrombin containingmodified mRNA and fibrinogen is loaded into a dual barreled syringe.Mice are injected subcutaneously with 50 ul of fibrinogen and 50 ul ofthrombin containing modified mRNA or they were injected with 50 ul ofPBS containing an equivalent dose of modified luciferase mRNA. A controlgroup of untreated mice is also evaluated. The mice are imaged atpredetermined intervals to determine the average total flux(photons/second).

C. Modified mRNA and Fibrinogen

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 33907; polyA tail of approximately 140 nucleotides not shown insequence; 5′cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with N1-methylpseudouridine isadded to the fibrinolysis inhibitor solution. The fibrinolysis inhibitorsolution is then used to reconstitute fibrinogen. Thrombin isreconstituted with the calcium chloride solution per the manufacturer'sinstructions. The reconstituted fibrinogen containing modified mRNA andthrombin is loaded into a dual barreled syringe. Mice are injectedsubcutaneously with 50 ul of thrombin and 50 ul of fibrinogen containingmodified mRNA or they were injected with 50 ul of PBS containing anequivalent dose of modified luciferase mRNA. A control group ofuntreated mice is also evaluated. The mice are imaged at predeterminedintervals to determine the average total flux (photons/second).

D. Lipid Nanoparticle Formulated Modified mRNA and Fibrinogen

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 33907; polyA tail of approximately 140 nucleotides not shown insequence; 5′cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with N1-methylpseudouridine isformulated in a lipid nanoparticle is added to the fibrinolysisinhibitor solution. The fibrinolysis inhibitor solution is then used toreconstitute fibrinogen. Thrombin is reconstituted with the calciumchloride solution per the manufacturer's instructions. The reconstitutedfibrinogen containing modified mRNA and thrombin is loaded into a dualbarreled syringe. Mice are injected subcutaneously with 50 ul ofthrombin and 50 ul of fibrinogen containing modified mRNA or they wereinjected with 50 ul of PBS containing an equivalent dose of modifiedluciferase mRNA. A control group of untreated mice is also evaluated.The mice are imaged at predetermined intervals to determine the averagetotal flux (photons/second).

E. Modified mRNA and Thrombin

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 33907; polyA tail of approximately 140 nucleotides not shown insequence; 5′cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with N1-methylpseudouridine isadded to the reconstituted thrombin after it is reconstituted with thecalcium chloride per the manufacturer's instructions. The fibrinolysisinhibitor solution is then used to reconstitute fibrinogen per themanufacturer's instructions. The reconstituted fibrinogen and thrombincontaining modified mRNA is loaded into a dual barreled syringe. Miceare injected subcutaneously with 50 ul of thrombin containing modifiedmRNA and 50 ul of fibrinogen or they were injected with 50 ul of PBScontaining an equivalent dose of modified luciferase mRNA. A controlgroup of untreated mice is also evaluated. The mice are imaged atpredetermined intervals to determine the average total flux(photons/second).

F. Lipid Nanoparticle Formulated Modified mRNA and Thrombin

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 33907; polyA tail of approximately 140 nucleotides not shown insequence; 5′cap, Cap1) fully modified with 5-methylcytosine andN1-methylpseudouridine or fully modified with N1-methyl-pseudouridine isformulated in a lipid nanoparticle is added to the reconstitutedthrombin after it is reconstituted with the calcium chloride per themanufacturer's instructions. The fibrinolysis inhibitor solution is thenused to reconstitute fibrinogen per the manufacturer's instructions. Thereconstituted fibrinogen and thrombin containing modified mRNA is loadedinto a dual barreled syringe. Mice are injected subcutaneously with 50ul of thrombin containing modified mRNA and 50 ul of fibrinogen or theywere injected with 50 ul of PBS containing an equivalent dose ofmodified luciferase mRNA. A control group of untreated mice is alsoevaluated. The mice are imaged at predetermined intervals to determinethe average total flux (photons/second).

Example 96 Cationic Lipid Formulation of 5-Methylcytosine andN1-Methyl-Pseudouridine Modified mRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fullymodified with 5-methylcytosine and N1-methyl-pseudouridine wasformulated in the cationic lipids described in Table 146. Theformulations were administered intravenously (I.V.), intramuscularly(I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 146 Cationic Lipid Formulations Formu- NPA- NPA- NPA- NPA-129-1111612-B lation 126-1 127-1 128-1 Lipid DLin- DLin- C12-200 DLinDMADODMA MC3- KC2- DMA DMA Lipid/ 20:1 20:1 20:1 20:1 20:1 mRNA ratio(wt/wt) Mean 122 nm 114 nm 153 nm 137 nm 223.2 nm Size PDI: 0.13 PDI:0.10 PDI: 0.17 PDI: 0.09 PDI: 0.142 Zeta at −1.4 mV −0.5 mV −1.4 mV  2.0mV  −3.09 mV pH 7.4 Encaps. 95% 77% 69% 80% 64% (RiboGr)

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 4.17E+05 p/s. The results of the imaging areshown in Table 147. In Table 147, “NT” means not tested.

TABLE 147 Flux DLin- DLin- MC3- KC2- Time DMA DMA C12-200 DLinDMA DODMARoute Point Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V. 2 hrs 1.92E+08 2.91E+08 1.08E+08 2.53E+07 8.40E+06 I.V.  8 hrs 1.47E+082.13E+08 3.72E+07 3.82E+07 5.62E+06 I.V. 24 hrs 1.32E+07 2.41E+075.35E+06 4.20E+06 8.97E+05 I.M.  2 hrs 8.29E+06 2.37E+07 1.80E+071.51E+06 NT I.M.  8 hrs 5.83E+07 2.12E+08 2.60E+07 1.99E+07 NT I.M. 24hrs 4.30E+06 2.64E+07 3.01E+06 9.46E+05 NT S.C.  2 hrs 1.90E+07 5.16E+078.91E+07 4.66E+06 9.61E+06 S.C.  8 hrs 7.74E+07 2.00E+08 4.58E+079.67E+07 1.90E+07 S.C. 24 hrs 7.49E+07 2.47E+07 6.96E+06 6.50E+061.28E+06

Example 97 Lipid Nanoparticle Intravenous Study

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) was formulated in alipid nanoparticle containing 50% DLin-MC3-DMA OR DLin-KC2-DMA asdescribed in Table 148, 38.5% cholesterol, 10% DSPC and 1.5% PEG. Theformulation was administered intravenously (I.V.) to Balb-C mice at adose of 0.5 mg/kg, 0.05 mg/kg, 0.005 mg/kg or 0.0005 mg/kg. Twentyminutes prior to imaging, mice were injected intraperitoneally with aD-luciferin solution at 150 mg/kg. Animals were then anesthetized andimages were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse.

TABLE 148 Formulations Formulation NPA-098-1 NPA-100-1 LipidDLin-KC2-DMA DLin-MC3-DMA Lipid/mRNA ratio (wt/wt) 20:1 20:1 Mean Size135 nm 152 nm PDI: 0.08 PDI: 0.08 Zeta at pH 7.4 −0.6 mV −1.2 mV Encaps.(RiboGr) 91% 94%

For DLin-KC2-DMA the mice were imaged at 2 hours, 8 hours, 24 hours, 72hours, 96 hours and 168 hours after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The background flux was about 3.66E+05 p/s.The results of the imaging are shown in Table 149. Organs were imaged at8 hours and the average total flux (photons/second) was measured for theliver, spleen, lung and kidney. A control for each organ was alsoanalyzed. The results are shown in Table 150. The peak signal for alldose levels was at 8 hours after administration. Also, distribution tothe various organs (liver, spleen, lung, and kidney) may be able to becontrolled by increasing or decreasing the LNP dose.

TABLE 149 Flux 0.5 mg/kg 0.05 mg/kg 0.005 mg/kg 0.0005 mg/kg Time PointFlux (p/s) Flux (p/s) Flux (p/s) Flux (p/s)  2 hrs 3.54E+08 1.75E+072.30E+06 4.09E+05  8 hrs 1.67E+09 1.71E+08 9.81E+06 7.84E+05 24 hrs2.05E+08 2.67E+07 2.49E+06 5.51E+05 72 hrs 8.17E+07 1.43E+07 1.01E+063.75E+05 96 hrs 4.10E+07 9.15E+06 9.58E+05 4.29E+05 168 hrs  3.42E+079.15E+06 1.47E+06 5.29E+05

TABLE 150 Organ Flux Liver Spleen Lung Kidney Flux (p/s) Flux (p/s) Flux(p/s) Flux (p/s)   0.5 mg/kg 1.42E+08 4.86E+07 1.90E+05 3.20E+05  0.05mg/kg 7.45E+06 4.62E+05 6.86E+04 9.11E+04  0.005 mg/kg 3.32E+05 2.97E+041.42E+04 1.15E+04 0.0005 mg/kg 2.34E+04 1.08E+04 1.87E+04 9.78E+03Untreated 1.88E+04 1.02E+04 1.41E+04 9.20E+03

For DLin-MC3-DMA the mice were imaged at 2 hours, 8 hours, 24 hours, 48hours, 72 hours and 144 hours after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The background flux was about 4.51E+05 p/s.The results of the imaging are shown in Table 151. Organs were imaged at8 hours and the average total flux (photons/second) was measured for theliver, spleen, lung and kidney. A control for each organ was alsoanalyzed. The results are shown in Table 152. The peak signal for alldose levels was at 8 hours after administration. Also, distribution tothe various organs (liver, spleen, lung, and kidney) may be able to becontrolled by increasing or decreasing the LNP dose.

TABLE 151 Flux 0.5 mg/kg 0.05 mg/kg 0.005 mg/kg 0.0005 mg/kg Time PointFlux (p/s) Flux (p/s) Flux (p/s) Flux (p/s)  2 hrs 1.23E+08 7.76E+067.66E+05 4.88E+05  8 hrs 1.05E+09 6.79E+07 2.75E+06 5.61E+05 24 hrs4.44E+07 1.00E+07 1.06E+06 5.71E+05 48 hrs 2.12E+07 4.27E+06 7.42E+054.84E+05 72 hrs 1.34E+07 5.84E+06 6.90E+05 4.38E+05 144 hrs  4.26E+062.25E+06 4.58E+05 3.99E+05

TABLE 152 Organ Flux Liver Spleen Lung Kidney Flux (p/s) Flux (p/s) Flux(p/s) Flux (p/s)   0.5 mg/kg 1.19E+08 9.66E+07 1.19E+06 1.85E+05  0.05mg/kg 1.10E+07 1.79E+06 7.23E+04 5.82E+04  0.005 mg/kg 3.58E+05 6.04E+041.33E+04 1.33E+04 0.0005 mg/kg 2.25E+04 1.88E+04 2.05E+04 1.65E+04Untreated 1.91E+04 1.66E+04 2.63E+04 2.14E+04

Example 98 Lipid Nanoparticle Subcutaneous Study

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) was formulated in alipid nanoparticle containing 50% DLin-KC2-DMA as described in Table153, 385% cholesterol, 10% DSPC and 1.5% PEG. The formulation wasadministered subcutaneously (S.C.) to Balb-C mice at a dose of 0.5mg/kg, 0.05 mg/kg or 0.005 mg/kg.

TABLE 153 DLin-KC2-DMA Formulation Formulation NPA-098-1 LipidDLin-KC2-DMA Lipid/mRNA ratio (wt/wt) 20:1 Mean Size 135 nm PDI: 0.08Zeta at pH 7.4 −0.6 mV Encaps. (RiboGr) 91%

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours, 24 hours, 48hours, 72 hours and 144 hours after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The lower limit of detection was about 3E+05p/s. The results of the imaging are shown in Table 154. Organs wereimaged at 8 hours and the average total flux (photons/second) wasmeasured for the liver, spleen, lung and kidney. A control for eachorgan was also analyzed. The results are shown in Table 155. The peaksignal for all dose levels was at 8 hours after administration. Also,distribution to the various organs (liver, spleen, lung, and kidney) maybe able to be controlled by increasing or decreasing the LNP dose. Athigh doses, the LNP formulations migrates outside of the subcutaneousinjection site, as high levels of luciferase expression are detected inthe liver, spleen, lung, and kidney.

TABLE 154 Flux 0.5 mg/kg 0.05 mg/kg 0.005 mg/kg Time Point Flux (p/s)Flux (p/s) Flux (p/s)  2 hrs 3.18E+07 7.46E+06 8.94E+05  8 hrs 5.15E+082.18E+08 1.34E+07 24 hrs 1.56E+08 5.30E+07 7.16E+06 48 hrs 5.22E+078.75E+06 9.06E+05 72 hrs 8.87E+06 1.50E+06 2.98E+05 144 hrs  4.55E+053.51E+05 2.87E+05

TABLE 155 Organ Flux Liver Spleen Lung Kidney Flux (p/s) Flux (p/s) Flux(p/s) Flux (p/s)  0.5 mg/kg 1.01E+07 7.43E+05 9.75E+04 1.75E+05  0.05mg/kg 1.61E+05 3.94E+04 4.04E+04 3.29E+04 0.005 mg/kg 2.84E+04 2.94E+042.42E+04 9.79E+04 Untreated 1.88E+04 1.02E+04 1.41E+04 9.20E+03

Example 99 Cationic Lipid Nanoparticle Subcutaneous Study

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) is formulated in alipid nanoparticle containing 50% DLin-MC3-DMA, 38.5% cholesterol, 10%DSPC and 1.5% PEG. The formulation is administered subcutaneously (S.C.)to Balb-C mice at a dose of 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg.

The mice are imaged at 2 hours, 8 hours, 24 hours, 48 hours, 72 hoursand 144 hours after dosing and the average total flux (photons/second)was measured for each route of administration and cationic lipidformulation. Organs are imaged at 8 hours and the average total flux(photons/second) is measured for the liver, spleen, lung and kidney. Acontrol for each organ is also analyzed.

Example 100 Luciferase Lipoplex Study

Lipoplexed luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907;polyA tail of approximately 140 nucleotides not shown in sequence;5′cap, Cap1) fully modified with 5-methylcytosine and pseudouridine(5mC/pU), fully modified with 5-methylcytosine andN1-methyl-pseudouridine (5mC/N1mpU) or modified where 25% of thecytosines replaced with 5-methylcytosine and 25% of the uridinesreplaced with 2-thiouridine (5mC/s2U). The formulation was administeredintravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) toBalb-C mice at a dose of 0.10 mg/kg.

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 8 hours, 24 hours and 48 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and chemical modification. Thebackground signal was about 3.91E+05 p/s. The results of the imaging areshown in Table 156. Organs were imaged at 6 hours and the average totalflux (photons/second) was measured for the liver, spleen, lung andkidney. A control for each organ was also analyzed. The results areshown in Table 157.

TABLE 156 Flux 5mC/pU 5mC/N1mpU 5mC/s2U Route Time Point Flux (p/s) Flux(p/s) Flux (p/s) I.V.  8 hrs 5.76E+06 1.78E+06 1.88E+06 I.V. 24 hrs1.02E+06 7.13E+05 5.28E+05 I.V. 48 hrs 4.53E+05 3.76E+05 4.14E+05 I.M. 8 hrs 1.90E+06 2.53E+06 1.29E+06 I.M. 24 hrs 9.33E+05 7.84E+05 6.48E+05I.M. 48 hrs 8.51E+05 6.59E+05 5.49E+05 S.C.  8 hrs 2.85E+06 6.48E+061.14E+06 S.C. 24 hrs 6.66E+05 7.15E+06 3.93E+05 S.C. 48 hrs 3.24E+053.20E+06 5.45E+05

TABLE 157 Organ Flux Spleen Kidney Inj. Site Route Chemistry Liver Flux(p/s) Flux (p/s) Lung Flux (p/s) Flux (p/s) Flux (p/s) I.V. 5mC/pU5.26E+05 2.04E+07 4.28E+06 1.77E+04 n/a I.V. 5mC/N1mpU 1.48E+05 5.00E+061.93E+06 1.77E+04 n/a I.V. 5mC/s2U 2.14E+04 3.29E+06 5.48E+05 2.16E+04n/a I.M. 5mC/pU 2.46E+04 1.38E+04 1.50E+04 1.44E+04 1.15E+06 I.M.5mC/N1mpU 1.72E+04 1.76E+04 1.99E+04 1.56E+04 1.20E+06 I.M. 5mC/s2U1.28E+04 1.36E+04 1.33E+04 1.07E+04 7.60E+05 S.C. 5mC/pU 1.55E+041.67E+04 1.45E+04 1.69E+04 4.46E+04 S.C. 5mC/N1mpU 1.20E+04 1.46E+041.38E+04 1.14E+04 8.29E+04 S.C. 5mC/s2U 1.22E+04 1.31E+04 1.45E+041.08E+04 5.62E+04 Untreated 2.59E+04 1.34E+04 1.26E+04 1.22E+04 n/a

Example 101 Cationic Lipid Formulation of Modified mRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1)modified where 25% of the cytosines replaced with 5-methylcytosine and25% of the uridines replaced with 2-thiouridine (5mC/s2U) was formulatedin the cationic lipids described in Table 158. The formulations wereadministered intravenously (I.V.), intramuscularly (I.M.) orsubcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 158 Cationic Lipid Formulations Formulation NPA- NPA- NPA- NPA-111612- 130-1 131-1 132-1 133-1 C Lipid DLin- DLin- C12-200 DLinDMADODMA MC3- KC2- DMA DMA Lipid/mRNA 20:1 20:1 20:1 20:1 20:1 ratio(wt/wt) Mean Size 120 nm 105 nm 122 nm 105 nm 221.3 nm PDI: 0.10 PDI:PDI: 0.13 PDI: 0.14 PDI: 0.11 0.063 Zeta at pH 0.2 mV −0.6 mV −0.5 mV−0.3 mV −3.10 mV 7.4 Encaps. 100% 100% 93% 93% 60% (RiboGr)

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 3.31E+05 p/s. The results of the imaging areshown in Table 159. In Table 159, “NT” means not tested. Untreated miceshowed an average flux of 3.14E+05 at 2 hours, 3.33E+05 at 8 hours and3.46E+05 at 24 hours. Peak expression was seen for all three routestested at 8 hours. DLin-KC2-DMA has better expression than DLin-MC3-DMAand DODMA showed expression for all routes evaluated.

TABLE 159 Flux DLin- DLin- MC3- KC2- Time DMA DMA C12-200 DLinDMA DODMARoute Point Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V. 2 hrs 9.88E+06 6.98E+07 9.18E+06 3.98E+06 5.79E+06 I.V.  8 hrs 1.21E+071.23E+08 1.02E+07 5.98E+06 6.14E+06 I.V. 24 hrs 2.02E+06 1.05E+071.25E+06 1.35E+06 5.72E+05 I.M.  2 hrs 6.72E+05 3.66E+06 3.25E+067.34E+05 4.42E+05 I.M.  8 hrs 7.78E+06 2.85E+07 4.29E+06 2.22E+061.38E+05 I.M. 24 hrs 4.22E+05 8.79E+05 5.95E+05 8.48E+05 4.80E+05 S.C. 2 hrs 2.37E+06 4.77E+06 4.44E+06 1.07E+06 1.05E+06 S.C.  8 hrs 3.65E+071.17E+08 3.71E+06 9.33E+06 2.57E+06 S.C. 24 hrs 4.47E+06 1.28E+076.39E+05 8.89E+05 4.27E+05

Example 102 Formulation of 5-Methylcytosine and N1-Methyl-pseudouridineModified mRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fullymodified with 5-methylcytosine and N1-methyl-pseudouridine wasformulated in PBS (pH of 7.4). The formulations were administeredintramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a doseof 2.5 mg/kg.

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 5 minutes, 30 minutes, 60minutes and 120 minutes after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The background flux was about 3.78E+05 p/s.The results of the imaging are shown in Table 160. Expression ofluciferase was already seen at 30 minutes with both routes of delivery.Peak expression from subcutaneous administration appears between 30 to60 minutes. Intramuscular expression was still increasing at 120minutes.

TABLE 160 Flux PBS (pH 7.4) Route Time Point Flux (p/s) I.M.  5 min4.38E+05 I.M. 30 min 1.09E+06 I.M. 60 min 1.18E+06 I.M. 120 min 2.86E+06 S.C.  5 min 4.19E+05 S.C. 30 min 6.38E+06 S.C. 60 min 5.61E+06S.C. 120 min  2.66E+06

Example 103 Intramuscular and Subcutaneous Administration of ChemicallyModified mRNA

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 140 nucleotides not shown in sequence; 5′cap,Cap1) fully modified with N4-acetylcytidine, fully modified with5-methoxyuridine, fully modified with N4-acetylcytidine andN1-methyl-pseudouridine or fully modified 5-methylcytosine and5-methoxyuridine formulated in PBS (pH 7.4) was administered to Balb-Cmice intramuscularly or subcutaneously at a dose of 2.5 mg/kg. Twentyminutes prior to imaging, mice were injected intraperitoneally with aD-luciferin solution at 150 mg/kg. Animals were then anesthetized andimages were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hours.The average total flux (photons/second) for intramuscular administrationis shown in Table 161 and the average total flux (photons/second) forsubcutaneous administration is shown in Table 162. The background signalwas 3.84E+05 (p/s). The peak expression for intramuscular administrationwas seen between 24 and 48 hours for all chemistry and expression wasstill detected at 120 hours. For subcutaneous delivery the peakexpression was seen at 2-8 hours and expression was detected at 72hours.

TABLE 161 Intramuscular Administration 2 hours 8 hours 24 hours Flux(p/s) Flux (p/s) Flux (p/s) N4-acetylcytidine 1.32E+07 2.15E+07 4.01E+075-methoxyuridine 4.93E+06 1.80E+07 4.53E+07 N4-acetylcytidine/ 2.02E+071.93E+07 1.63E+08 N1-methylpseudouridine 5-methylcytosine/5- 6.79E+064.55E+07 3 .44E+07 methoxyuridine

TABLE 162 Subcutaneous Administration 2 hours 8 hours 24 hours Flux(p/s) Flux (p/s) Flux (p/s) N4-acetylcytidine 3.07E+07 1.23E+07 1.28E+075-methoxyuridine 7.10E+06 9.38E+06 1.32E+07 N4-acetylcytidine/ 7.12E+063 .07E+06 1.03E+07 N1-methylpseudouridine 5-methylcytosine/5- 7.15E+061.25E+07 1.11E+07 methoxyuridine

Example 104 In Vivo Study

Luciferase modified mRNA containing at least one chemical modificationis formulated as a lipid nanoparticle (LNP) using the syringe pumpmethod and characterized by particle size, zeta potential, andencapsulation.

As outlined in Table 163, the luciferase LNP formulation is administeredto Balb-C mice intramuscularly (I.M.), intravenously (I.V.) andsubcutaneously (S.C.). As a control luciferase modified RNA formulatedin PBS is administered intravenously to mice.

TABLE 163 Luciferase Formulations Con- Injection Amount of centrationVolume modified Dose Formulation Vehicle Route (mg/ml) (ul) RNA (ug)(mg/kg) Luc-LNP PBS S.C. 0.2000 50 10 0.5000 Luc-LNP PBS S.C. 0.0200 501 0.0500 Luc-LNP PBS S.C. 0.0020 50 0.1 0.0050 Luc-LNP PBS S.C. 0.000250 0.01 0.0005 Luc-LNP PBS I.V. 0.2000 50 10 0.5000 Luc-LNP PBS I.V.0.0200 50 1 0.0500 Luc-LNP PBS I.V. 0.0020 50 0.1 0.0050 Luc-LNP PBSI.V. 0.0002 50 0.01 0.0005 Luc-LNP PBS I.M. 0.2000 50 10 0.5000 Luc-LNPPBS I.M. 0.0200 50 1 0.0500 Luc-LNP PBS I.M. 0.0020 50 0.1 0.0050Luc-LNP PBS I.M. 0.0002 50 0.01 0.0005 Luc-PBS PBS I.V. 0.20 50 10 0.50

The mice are imaged at 2, 8, 24, 48, 120 and 192 hours to determine thebioluminescence (measured as total flux (photons/second) of the entiremouse). At 8 hours or 192 hours the liver, spleen, kidney and injectionsite for subcutaneous and intramuscular administration are imaged todetermine the bioluminescence.

Example 105 Cationic Lipid Formulation Studies of Chemically ModifiedmRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU), pseudouridine(pU) or N1-methyl-pseudouridine (N1mpU) was formulated in the cationiclipids described in Table 164. The formulations were administeredintravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) toBalb-C mice at a dose of 0.05 mg/kg.

TABLE 164 Cationic Lipid Formulations Formulation NPA- NPA- NPA- NPA-111612- 137-1 134-1 135-1 136-1 A Lipid DLin- DLin- DLin-KC2- C12-200DODMA MC3- MC3- DMA DMA DMA Lipid/mRNA 20:1 20:1 20:1 20:1 20:1 ratio(wt/wt) Mean Size 111 nm 104 nm 95 nm 143 nm 223.2 nm PDI: 0.15 PDI:0.13 PDI: 0.11 PDI: PDI: 0.12 0.142 Zeta at −4.1 mV −1.9 mV −1.0 mV 0.2mV −3.09 mV pH 7.4 Encaps. 97% 100% 100% 78% 64% (RiboGr) Chemistry pUN1mpU N1mpU N1mpU 5mC/pU

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 4.11E+05 p/s. The results of the imaging areshown in Table 165. Peak expression was seen for all three routes testedat 8 hours.

TABLE 165 Flux DLin- DLin- DLin- MC3- MC3- KC2- DMA DMA DMA C12-200DODMA Time (pU) (N1mpU) (N1mpU) (N1mpU) (5mC/pU) Route Point Flux (p/s)Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V.  2 hrs 3.21E+081.24E+09 1.01E+09 9.00E+08 3.90E+07 I.V.  8 hrs 1.60E+09 3.22E+092.38E+09 1.11E+09 1.17E+07 I.V. 24 hrs 1.41E+08 3.68E+08 3.93E+088.06E+07 1.11E+07 I.M.  2 hrs 2.09E+07 3.29E+07 8.32E+07 9.43E+074.66E+06 I.M.  8 hrs 2.16E+08 6.14E+08 1.00E+09 8.77E+07 7.05E+06 I.M.24 hrs 1.23E+07 1.40E+08 5.09E+08 1.36E+07 1.14E+06 S.C.  2 hrs 2.32E+073.60E+07 2.14E+08 1.01E+08 3.11E+07 S.C.  8 hrs 5.55E+08 9.80E+084.93E+09 1.01E+09 8.04E+07 S.C. 24 hrs 1.81E+08 2.74E+08 2.12E+094.74E+07 1.34E+07

Example 106 Studies of Chemical Modified mRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fullymodified with N4-acetylcytidine (N4-acetyl), fully modified with5-methoxyuridine (5meth), fully modified with N4-acetylcytidine andN1-methyl-pseudouridine (N4-acetyl/N1mpU) or fully modified with5-methylcytosine and 5-methoxyuridine (5mC/5-meth) was formulated inDLin-MC3-DMA as described in Table 166.

The formulations were administered intravenously (I.V.), intramuscularly(I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 166 Cationic Lipid Formulations Formulation NPA-141-1 NPA-142-1NPA-143-1 NPA-144-1 Lipid DLin- DLin- DLin- DLin- MC3-DMA MC3-DMAMC3-DMA MC3-DMA Lipid/mRNA 20:1 20:1 20:1 20:1 ratio (wt/wt) Mean Size138 nm 116 nm 144 nm 131 nm PDI: 0.16 PDI: 0.15 PDI: 0.15 PDI: 0.15 Zetaat pH 7.4 −2.8 mV −2.8 mV −4.3 mV −5.0 mV Encaps. 97% 100% 75% 72%(RiboGr) Chemistry N4-acetyl 5meth N4-acetyl/ 5mC/5-meth N1mpU

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 6 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 2.70E+05 p/s. The results of the imaging areshown in Table 167.

TABLE 167 Flux N4-acetyl/ 5mC/5- N4-acetyl 5meth N1mpU meth Route TimePoint Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V.  2 hrs 9.17E+073.19E+06 4.21E+07 1.88E+06 I.V.  6 hrs 7.70E+08 9.28E+06 2.34E+087.75E+06 I.V. 24 hrs 6.84E+07 1.04E+06 3.55E+07 3.21E+06 I.M.  2 hrs8.59E+06 7.86E+05 5.30E+06 5.11E+05 I.M.  6 hrs 1.27E+08 8.88E+063.82E+07 3.17E+06 I.M. 24 hrs 4.46E+07 1.38E+06 2.00E+07 1.39E+06 S.C. 2 hrs 1.83E+07 9.67E+05 4.45E+06 1.01E+06 S.C.  6 hrs 2.89E+08 1.78E+078.91E+07 1.29E+07 S.C. 24 hrs 6.09E+07 6.40E+06 2.08E+08 6.63E+06

Example 107 Lipid Nanoparticle Containing a Plurality of Modified mRNAs

EPO mRNA (mRNA sequence shown in SEQ ID NO: 33900; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with 5-methylcytosine and N1-methyl-pseudouridine), G-CSF mRNA(mRNA sequence shown in SEQ ID NO: 33897; polyA tail of approximately140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with5-methylcytosine and N1-methyl-pseudouridine) and Factor IX mRNA (mRNAsequence shown in SEQ ID NO: 33901; polyA tail of approximately 140nucleotides not shown in sequence; 5′cap, Cap1; fully modified with5-methylcytosine and N1-methylpseudouridine), is formulated inDLin-MC3-DMA as described in Table 168. The formulations areadministered intravenously (I.V.), intramuscularly (I.M.) orsubcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg. ControlLNP formulations containing only one mRNA are also administered at anequivalent dose.

TABLE 168 DLin-MC3-DMA Formulation Formulation NPA-157-1 LipidDLin-MC3-DMA Lipid/mRNA 20:1 ratio (wt/wt) Mean Size  89 nm PDI: 0.08Zeta at pH 7.4 1.1 mV Encaps. 97% (RiboGr)

Serum is collected from the mice at 8 hours, 24 hours, 72 hours and/or 7days after administration of the formulation. The serum is analyzed byELISA to determine the protein expression of EPO, G-CSF, and Factor IX.

Example 108 Cationic Lipid Formulation Studies of 5-Methylcytosine andN1-Methyl-pseudouridine Modified mRNA

EPO mRNA (mRNA sequence shown in SEQ ID NO: 33900; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with 5-methylcytosine and N1-methyl-pseudouridine) or G-CSFmRNA (mRNA sequence shown in SEQ ID NO: 33897; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fullymodified with 5-methylcytosine and N1-methylpseudouridine) is formulatedin DLin-MC3-DMA and DLin-KC2-DMA as described in Table 169. Theformulations are administered intravenously (I.V), intramuscularly(I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 169 DLin-MC3-DMA and DLin-KC2-DMA Formulations FormulationNPA-147-1 NPA-148-1 NPA-150-1 NPA-151-1 mRNA EPO EPO G-CSF G-CSF LipidDLin-MC3- DLin-KC2- DLin-MC3- DLin-KC2- DMA DMA DMA DMA Lipid/mRNA 20:120:1 20:1 20:1 ratio (wt/wt) Mean Size  117 nm  82 nm  119 nm  88 nmPDI: 0.14 PDI: 0.08 PDI: 0.13 PDI: 0.08 Zeta at pH 7.4 −1.7 mV 0.6 mV 3.6 mV 2.2 mV Encaps. (RiboGr) 100% 96% 100% 100%

Serum is collected from the mice at 8 hours, 24 hours, 72 hours and/or 7days after administration of the formulation. The serum is analyzed byELISA to determine the protein expression of EPO and G-CSF.

Example 109 In Vitro VEGF PBMC Study

500 ng of VEGF mRNA (mRNA sequence shown in SEQ ID NO: 33910 polyA tailof approximately 160 nucleotides not shown in sequence; 5′cap, Cap1)fully modified with 5-methylcytosine and pseudouridine (VEGF 5mC/pU),fully modified with 5-methylcytosine and N1-methyl-pseudouridine (VEGF5mC/N1mpU) or unmodified (VEGF unmod) was transfected with 0.4 uL ofLipofectamine 2000 into peripheral blood mononuclear cells (PBMC) fromthree normal blood donors (D1, D2, and D3). Cells were also untreatedfor each donor as a control. The supernatant was harvested and run byELISA 22 hours after transfection to determine the protein expressionand cytokine induction. The expression of VEGF and IFN-alpha inductionis shown in Table 170.

TABLE 170 Protein and Cytokine levels VEGF Expression IFN-alphaInduction (pg/ml) (pg/ml) D1 D2 D3 D1 D2 D3 VEGF unmod 2 0 0 5400 35374946 VEGF 5mC/pU 424 871 429 145 294 106 VEGF 5mC/N1mpU 5088 10331 6183205 165 6

Example 110 In Vitro Expression of Modified mRNA

HEK293 cells were transfected with VEGF-A modified mRNA (mRNA sequenceshown in SEQ ID NO: 33910; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) or EPO modified mRNA (mRNA sequenceshown in SEQ ID NO: 33900; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosineand pseudouridine), HeLa cells were forward transfected withTransforming growth factor beta (TGF-beta) modified mRNA (mRNA sequenceshown in SEQ ID NO: 33911; poly A tail of approximately 160 nucleotidesnot shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosineand pseudouridine) and HepG2 cells were transfected withbactericidal/permeability-increasing protein (rBPI-21) modified mRNA(mRNA sequence shown in SEQ ID NO: 33912; poly A tail of approximately160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with5-methylcytosine and pseudouridine) which had been complexed withLipofectamine 2000 from Invitrogen (Carlsbad, Calif.) at theconcentrations shown in Table 171, 172 and 173 using the proceduresdescribed herein. The protein expression was detected by ELISA and theprotein (pg/ml) is also shown in Table 171, 172 and 173. In Table 171,“>” means greater than. For TGF-beta a control of untreated cells and amock transfection of Lipofectamine 2000 was also tested.

TABLE 171 EPO Protein Expression Amount 5 ng 1 ng 200 pg 40 pg 8 pg 1.6pg 320 fg 64 fg Trans- fected Protein >2000 609.486 114.676 0 0 0 0 0(pg/ml)

TABLE 172 TGF-beta Protein Expression Amount 750 ng 250 ng 83 ng MockUntreated Transfected Protein 5058 4325 3210 2 0 (pg/ml)

TABLE 173 rBPI-21 Protein Expression Amount 2 ug 400 ng 80 ng 16 ng 3.2ng 640 pg 128 26 Trans- pg pg fected Protein 20.683 9.269 4.768 0 0 0 00 (pg/ml)

Example 111 Bicistronic Modified mRNA

Human embryonic kidney epithelial (HEK293) were seeded on 96-well plates(Greiner Bio-one GmbH, Frickenhausen, Germany) HEK293 were seeded at adensity of 30,000 in 100 μl cell culture medium (DMEM, 10% FCS, adding 2mM L-Glutamine, 1 mM Sodiumpyruvate and 1× non-essential amino acids(Biochrom AG, Berlin, Germany) and 1.2 mg/ml Sodiumbicarbonate(Sigma-Aldrich, Munich, Germany)) 75 ng of the bi-cistronic modifiedmRNA (mCherry-2A-GFP) (SEQ ID NO: 33913; polyA tail of approximately 160nucleotides not shown in sequence; 5′cap, Cap1; fully modified with5-methylcytosine and pseudouridine), mCherry modified mRNA (mRNA SEQ IDNO: 33898; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) or green fluorescent protein (GFP) modified mRNA (mRNAsequence shown in SEQ ID NO: 33909; polyA tail of approximately 160nucleotides not shown in sequence; 5′cap, Cap1; fully modified with5-methylcytosine and pseudouridine) were added after seeding the cellsand incubated. A control of untreated cells was also evaluated.mCherry-2A-GFP refers to a modified mRNA sequence comprising the codingregion of mCherry, the 2A peptide and the coding region of GFP.

Cells were harvested by transferring the culture media supernatants to a96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg,Germany). Cells were trypsinized with ½ volume Trypsin/EDTA (BiochromAG, Berlin, Germany), pooled with respective supernatants and fixed byadding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5%formaldehyde (Merck, Darmstadt, Germany). Samples then were submitted toa flow cytometer measurement with a 532 nm excitation laser and the610/20 filter for PE-Texas Red in a LSRII cytometer (Beckton DickinsonGmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI) of allevents is shown in Table 174. Cells transfected with the bi-cistronicmodified mRNA were able to express both mCherry and GFP.

TABLE 174 MFI of Modified mRNA Modified mRNA mCherry MFI GFP MFI mCherry17746 427 GFP 427 20019 mCherry-2A-GFP 5742 6783 Untreated 427 219

Example 112 Cationic Lipid Formulation Studies of 5-Methylcytosine andN1-Methylpseudouridine Modified mRNA to Produce an Antibody

Herceptin heavy chain (HC) modified mRNA (mRNA sequence shown in SEQ IDNO: 33914; polyA tail of approximately 140 nucleotides not shown insequence; 5′cap, Cap1; fully modified with 5-methylcytosine andN1-methylpseudouridine) and Herceptin light chain (LC) modified mRNA(mRNA sequence shown in SEQ ID NO: 33915; polyA tail of approximately140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with5-methylcytosine and N1-methylpseudouridine) are formulated inDLin-MC3-DMA and DLin-KC2-DMA as described in Table 175. Theformulations are administered intravenously (I.V) to Balb-C mice at adose of 0.500, 0.050, and 0.005 mg/kg.

TABLE 175 DLin-MC3-DMA and DLin-KC2-DMA Formulations FormulationNPA-158-1 NPA-159-1 Herceptin HC:LC 2:1 2:1 Ratio (wt/wt) LipidDLin-MC3- DLin-KC2- DMA DMA Lipid/Total mRNA 20:1 20:1 ratio (wt/wt)Mean Size  129 nm  100 nm PDI: 0.14 PDI: 0.10 Zeta at pH 7.4  0.9 mV 1.9 mV Encaps. (RiboGr) 100% 100%

Serum was collected from the mice at 8 hours, 24 hours, 72 hours and/or7 days after administration of the formulation. The serum was analyzedby ELISA to determine the protein expression of Herceptin.

Example 113 Directed SAR of Pseudouridine and N1-Methyl-Pseudouridine

With the recent focus on the pyrimidine nucleoside pseudouridine, aseries of structure-activity studies were designed to investigate mRNAcontaining modifications to pseudouridine or N1-methyl-pseudouridine.

The study was designed to explore the effect of chain length, increasedlipophilicity, presence of ring structures, and alteration ofhydrophobic or hydrophilic interactions when modifications were made atthe N1 position, C6 position, the 2-position, the 4-position and on thephosphate backbone. Stability is also investigated.

To this end, modifications involving alkylation, cycloalkylation,alkyl-cycloalkylation, arylation, alkyl-arylation, alkylation moietieswith amino groups, alkylation moieties with carboxylic acid groups, andalkylation moieties containing amino acid charged moieties areinvestigated. The degree of alkylation is generally C₁-C₆. Examples ofthe chemistry modifications include those listed in Table 176 and Table177.

TABLE 176 Pseudouridine and N1-methyl-pseudouridine SAR CompoundNaturally Chemistry Modification # occuring N1-ModificationsN1-Ethyl-pseudo-UTP 1 N N1-Propyl-pseudo-UTP 2 NN1-iso-propyl-pseudo-UTP 3 N N1-(2,2,2-Trifluoroethyl)-pseudo-UTP 4 NN1-Cyclopropyl-pseudo-UTP 5 N N1-Cyclopropylmethyl-pseudo-UTP 6 NN1-Phenyl-pseudo-UTP 7 N N1-Benzyl-pseudo-UTP 8 NN1-Aminomethyl-pseudo-UTP 9 N P seudo-UTP-N1-2-ethanoic acid 10 N N1-(3-Amino-3-carboxypropyl)pseudo-UTP 11 NN1-Methyl-3-(3-amino-3-carboxypropyl)pseudo- 12 Y UTP C-6 Modifications6-Methyl-pseudo-UTP 13 N 6-Trifluoromethyl-pseudo-UTP 14 N6-Methoxy-pseudo-UTP 15 N 6-Phenyl-pseudo-UTP 16 N 6-Iodo-pseudo-UTP 17N 6-Bromo-pseudo-UTP 18 N 6-Chloro-pseudo-UTP 19 N 6-Fluoro-pseudo-UTP20 N 2-or 4-position Modifications 4-Thio-pseudo-UTP 21 N2-Thio-pseudo-UTP 22 N Phosphate backbone ModificationsAlpha-thio-pseudo-UTP 23 N N1-Me-alpha-thio-pseudo-UTP 24 N

TABLE 177 Pseudouridine and N1-methyl-pseudouridine SAR CompoundNaturally Chemistry Modification # occurring N1-Methyl-pseudo-UTP 1 YN1-Butyl-pseudo-UTP 2 N N1-tert-Butyl-pseudo-UTP 3 NN1-Pentyl-pseudo-UTP 4 N N1-Hexyl-pseudo-UTP 5 NN1-Trifluoromethyl-pseudo-UTP 6 Y N1-Cyclobutyl-pseudo-UTP 7 NN1-Cyclopentyl-pseudo-UTP 8 N N1-Cyclohexyl-pseudo-UTP 9 NN1-Cycloheptyl-pseudo-UTP 10 N N1-Cyclooctyl-pseudo-UTP 11 NN1-Cyclobutylmethyl-pseudo-UTP 12 N N1-Cyclopentylmethyl-pseudo-UTP 13 NN1-Cyclohexylmethyl-pseudo-UTP 14 N N1-Cycloheptylmethyl-pseudo-UTP 15 NN1-Cyclooctylmethyl-pseudo-UTP 16 N N1-p-tolyl-pseudo-UTP 17 NN1-(2,4,6-Trimethyl-phenyl)pseudo-UTP 18 NN1-(4-Methoxy-phenyl)pseudo-UTP 19 N N1-(4-Amino-phenyl)pseudo-UTP 20 NN1(4-Nitro-phenyl)pseudo-UTP 21 N Pseudo-UTP-N1-p-benzoic acid 22 NN1-(4-Methyl-benzyl)pseudo-UTP 24 NN1-(2,4,6-Trimethyl-benzyl)pseudo-UTP 23 NN1-(4-Methoxy-benzyl)pseudo-UTP 25 N N1-(4-Amino-benzyl)pseudo-UTP 26 NN1-(4-Nitro-benzyl)pseudo-UTP 27 N Pseudo-UTP-N1-methyl-p-benzoic acid28 N N1-(2-Amino-ethyl)pseudo-UTP 29 N N1-(3-Amino-propyl)pseudo-UTP 30N N1-(4-Amino-butyl)pseudo-UTP 31 N N1-(5-Amino-pentyl)pseudo-UTP 32 NN1-(6-Amino-hexyl)pseudo-UTP 33 N Pseudo-UTP-N1-3-propionic acid 34 NPseudo-UTP-N1-4-butanoic acid 35 N Pseudo-UTP-N1-5-pentanoic acid 36 NPseudo-UTP-N1-6-hexanoic acid 37 N Pseudo-UTP-N1-7-heptanoic acid 38 NN1-(2-Amino-2-carboxyethyl)pseudo-UTP 39 NN1-(4-Amino-4-carboxybutyl)pseudo-UTP 40 N N3-Alkyl-pseudo-UTP 41 N6-Ethyl-pseudo-UTP 42 N 6-Propyl-pseudo-UTP 43 N 6-iso-Propyl-pseudo-UTP44 N 6-Butyl-pseudo-UTP 45 N 6-tert-Butyl-pseudo-UTP 46 N6-(2,2,2-Trifluoroethyl)-pseudo-UTP 47 N 6-Ethoxy-pseudo-UTP 48 N6-Trifluoromethoxy-pseudo-UTP 49 N 6-Phenyl-pseudo-UTP 50 N6-(Substituted-Phenyl)-pseudo-UTP 51 N 6-Cyano-pseudo-UTP 52 N6-Azido-pseudo-UTP 53 N 6-Amino-pseudo-UTP 54 N6-Ethylcarboxylate-pseudo-UTP 54b N 6-Hydroxy-pseudo-UTP 55 N6-Methylamino-pseudo-UTP 55b N 6-Dimethylamino-pseudo-UTP 57 N6-Hydroxyamino-pseudo-UTP 59 N 6-Formyl-pseudo-UTP 60 N6-(4-Morpholino)-pseudo-UTP 61 N 6-(4-Thiomorpholino)-pseudo-UTP 62 NN1-Me-4-thio-pseudo-UTP 63 N N1-Me-2-thio-pseudo-UTP 64 N1,6-Dimethyl-pseudo-UTP 65 N 1-Methyl-6-trifluoromethyl-pseudo-UTP 66 N1-Methyl-6-ethyl-pseudo-UTP 67 N 1-Methyl-6-propyl-pseudo-UTP 68 N1-Methyl-6-iso-propyl-pseudo-UTP 69 N 1-Methyl-6-butyl-pseudo-UTP 70 N1-Methyl-6-tert-butyl-pseudo-UTP 71 N1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP 72 N1-Methyl-6-iodo-pseudo-UTP 73 N 1-Methyl-6-bromo-pseudo-UTP 74 N1-Methyl-6-chloro-pseudo-UTP 75 N 1-Methyl-6-fluoro-pseudo-UTP 76 N1-Methyl-6-methoxy-pseudo-UTP 77 N 1-Methyl-6-ethoxy-pseudo-UTP 78 N1-Methyl-6-trifluoromethoxy-pseudo-UTP 79 N 1-Methyl-6-phenyl-pseudo-UTP80 N 1-Methyl-6-(substituted phenyl)pseudo-UTP 81 N1-Methyl-6-cyano-pseudo-UTP 82 N 1-Methyl-6-azido-pseudo-UTP 83 N1-Methyl-6-amino-pseudo-UTP 84 N 1-Methyl-6-ethylcarboxylate-pseudo-UTP85 N 1-Methyl-6-hydroxy-pseudo-UTP 86 N1-Methyl-6-methylamino-pseudo-UTP 87 N1-Methyl-6-dimethylamino-pseudo-UTP 88 N1-Methyl-6-hydroxyamino-pseudo-UTP 89 N 1-Methyl-6-formyl-pseudo-UTP 90N 1-Methyl-6-(4-morpholino)-pseudo-UTP 91 N1-Methyl-6-(4-thiomorpholino)-pseudo-UTP 92 N 1-Alkyl-6-vinyl-pseudo-UTP93 N 1-Alkyl-6-allyl-pseudo-UTP 94 N 1-Alkyl-6-homoallyl-pseudo-UTP 95 N1-Alkyl-6-ethynyl-pseudo-UTP 96 N 1-Alkyl-6-(2-propynyl)-pseudo-UTP 97 N1-Alkyl-6-(1-propynyl)-pseudo-UTP 98 N

Example 114 Incorporation of Naturally and Non-Naturally OccurringNucleosides

Naturally and non-naturally occurring nucleosides are incorporated intomRNA encoding a polypeptide of interest. Examples of these are given inTables 178 and 179. Certain commercially available nucleosidetriphosphates (NTPs) are investigated in the polynucleotides of theinvention. A selection of these are given in Table 179. The resultantmRNA are then examined for their ability to produce protein, inducecytokines, and/or produce a therapeutic outcome.

TABLE 178 Naturally and non-naturally occurring nucleosides CompoundNaturally Chemistry Modification # occuring N4-Methyl-Cytosine 1 YN4,N4-Dimethyl-2′-OMe-Cytosine 2 Y 5-Oxyacetic acid-methyl ester-Uridine3 Y N3-Methyl-pseudo-Uridine 4 Y 5-Hydroxymethyl-Cytosine 5 Y5-Trifluoromethyl-Cytosine 6 N 5-Trifluoromethyl-Uridine 7 N5-Methyl-amino-methyl-Uridine 8 Y 5-Carboxy-methyl-amino-methyl-Uridine9 Y 5-Carboxymethylaminomethyl-2′-OMe-Uridine 10 Y5-Carboxymethylaminomethyl-2-thio-Uridine 11 Y5-Methylaminomethyl-2-thio-Uridine 12 Y5-Methoxy-carbonyl-methyl-Uridine 13 Y5-Methoxy-carbonyl-methyl-2′-OMe-Uridine 14 Y 5-Oxyacetic acid-Uridine15 Y 3-(3-Amino-3-carboxypropyl)-Uridine 16 Y5-(carboxyhydroxymethyl)uridine methyl ester 17 Y5-(carboxyhydroxymethyl)uridine 18 Y

TABLE 179 Non-naturally occurring nucleoside triphosphates CompoundNaturally Chemistry Modification # occuring N1-Me-GTP 1 N2′-OMe-2-Amino-ATP 2 N 2′-OMe-pseudo-UTP 3 Y 2′-OMe-6-Me-UTP 4 N2′-Azido-2′-deoxy-ATP 5 N 2′-Azido-2′-deoxy-GTP 6 N2′-Azido-2′-deoxy-UTP 7 N 2′-Azido-2′-deoxy-CTP 8 N2′-Amino-2′-deoxy-ATP 9 N 2′-Amino-2′-deoxy-GTP 10 N2′-Amino-2′-deoxy-UTP 11 N 2′-Amino-2′-deoxy-CTP 12 N 2-Amino-ATP 13 N8-Aza-ATP 14 N Xanthosine-5′-TP 15 N 5-Bromo-CTP 16 N2′-F-5-Methyl-2′-deoxy-UTP 17 N 5-Aminoallyl-CTP 18 N2-Amino-riboside-TP 19 N

Example 115 Incorporation of Modifications to the Nucleobase andCarbohydrate (Sugar)

Naturally and non-naturally occurring nucleosides are incorporated intomRNA encoding a polypeptide of interest. Commercially availablenucleosides and NTPs having modifications to both the nucleobase andcarbohydrate (sugar) are examined for their ability to be incorporatedinto mRNA and to produce protein, induce cytokines, and/or produce atherapeutic outcome. Examples of these nucleosides are given in Tables180 and 181.

TABLE 180 Combination modifications Compound Chemistry Modification #5-iodo-2′-fluoro-deoxyuridine 1 5-iodo-cytidine 6 2′-bromo-deoxyuridine7 8-bromo-adenosine 8 8-bromo-guanosine 9 2,2′-anhydro-cytidinehydrochloride 10 2,2′-anhydro-uridine 11 2′-Azido-deoxyuridine 122-amino-adenosine 13 N4-Benzoyl-cytidine 14 N4-Amino-cytidine 152′-O-Methyl-N4-Acetyl-cytidine 16 2′Fluoro-N4-Acetyl-cytidine 172′Fluor-N4-Bz-cytidine 18 2′O-methyl-N4-Bz-cytidine 192′O-methyl-N6-Bz-deoxyadenosine 20 2′Fluoro-N6-Bz-deoxyadenosine 21N2-isobutyl-guanosine 22 2′Fluro-N2-isobutyl-guanosine 232′O-methyl-N2-isobutyl-guanosine 24

TABLE 181 Naturally occuring combinations Compound Naturally Name #occurring 5-Methoxycarbonylmethyl-2-thiouridine TP 1 Y5-Methylaminomethyl-2-thiouridine TP 2 Y 5-Carbamoylmethyluridine TP 3 Y5-Carbamoylmethyl-2′-O-methyluridine TP 4 Y1-Methyl-3-(3-amino-3-carboxypropyl) 5 Y pseudouridine TP5-Methylaminomethyl-2-selenouridine TP 6 Y 5-Carboxymethyluridine TP 7 Y5-Methyldihydrouridine TP 8 Y lysidine TP 9 Y 5-Taurinomethyluridine TP10 Y 5-Taurinomethyl-2-thiouridine TP 11 Y5-(iso-Pentenylaminomethyl)uridine TP 12 Y5-(iso-Pentenylaminomethyl)-2-thiouridine TP 13 Y5-(iso-Pentenylaminomethyl)-2′-O- 14 Y methyluridine TPN4-Acetyl-2′-O-methylcytidine TP 15 Y N4,2′-O-Dimethylcytidine TP 16 Y5-Formyl-2′-O-methylcytidine TP 17 Y 2′-O-Methylpseudouridine TP 18 Y2-Thio-2′-O-methyluridine TP 19 Y 3,2′-O-Dimethyluridine TP 20 Y

In the tables “UTP” stands for uridine triphosphate, “GTP” stands forguanosine triphosphate, “ATP” stands for adenosine triphosphate, “CTP”stands for cytosine triphosphate, “TP” stands for triphosphate and “Bz”stands for benzyl.

Example 116 Dose Response and Injection Site Selection and Timing

To determine the dose response trends, impact of injection site andimpact of injection timing, studies are performed following the protocoloutlined in Example 35. In these studies, varied doses of 1 ug, 5 ug, 10ug, 25 ug, 50 ug, and values in between are used to determine doseresponse outcomes. Split dosing for a 100 ug total dose includes threeor six doses of 1.6 ug, 4.2 ug, 8.3 ug, 16.6 ug, or values and totaldoses equal to administration of the total dose selected.

Injection sites are chosen from the limbs or any body surface presentingenough area suitable for injection. This may also include a selection ofinjection depth to target the dermis (Intradermal), epidermis(Epidermal), subcutaneous tissue (SC) or muscle (IM). Injection anglewill vary based on targeted delivery site with injections targeting theintradermal site to be 10-15 degree angles from the plane of the surfaceof the skin, between 20-45 degrees from the plane of the surface of theskin for subcutaneous injections and angles of between 60-90 degrees forinjections substantially into the muscle.

Example 117 Intranasal Lung Delivery of 1-methylpseudouridine or5-methylcytosine and 1-methylpseudouridine Modified Luciferase mRNAFormulated in Cationic Lipid Nanoparticle

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 140 nucleotides not shown in sequence; 5′cap,Cap1) fully modified with 1-methylpseudouridine (Luc-G5-LNP-KC2) orfully modified with 1-methylpseudouridine and 5-methylcytosine(Luc-G2-LNP-KC2) were formulated in the cationic Lipid Nanoparticle(LNP-KC2). The formulations were administered intranasally (I.N.) toBalb-C mice at a dose of 0.3 mg/kg. Twenty minutes prior to imaging,mice were injected intraperitoneally with a D-luciferin solution at 150mg/kg. Animals were then anesthetized and images were acquired with anIVIS Lumina II imaging system (Perkin Elmer). Bioluminescence wasmeasured as total flux (photons/second) of the entire mouse. The micewere imaged at 2 hours, 8 hours, 48, and 72 hours after dosing and theaverage total flux (photons/second) was measured. The background fluxwas about 6.3+05 p/s. The results of the imaging of Luc-G5-LNP-KC2 orLuc-G5-LNP-KC2 vs. Vehicle are shown in the Table 182, “NT” means nottested.

TABLE 182 Luciferase expression after intranasal dosing Luc-G5-LNP- TimeKC2 Luc-G2-LNP-KC2 Vehicle (PBS) Route Point Flux (p/s) Flux (p/s) Flux(p/s) I.N.  2 hrs 1.53E+06 5.81E+05 3.87E+05 I.N.  8 hrs 1.09E+079.35E+05 5.57E+05 I.N. 24 hrs 5.93E+06 5.33E+05 4.89E+05 I.N. 28 hrs1.22E+06 7.63E+05 8.81E+05 I.N. 72 hrs 8.43E+05 NT 8.17E+05

Example 118 Intranasal Lung Delivery of 1-methylpseudouridine or5-methylcytosine and 1-methylpseudouridine Modified Luciferase mRNAlipoplexed in Lipofectamine 2000

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 140 nucleotides not shown in sequence; 5′cap,Cap1) fully modified with 1-methylpseudouridine (Luc-G5-Lipoplex) orfully modified with 1-methylpseudouridine and 5-methylcytosine(Luc-G2-Lipoplex) were lipoplexed in two steps, first step was thedilution of 16.6 ul from 3 mg/ml of G5 or G2 modified luciferase mRNA in108.5 ul DMEM and the second step was the dilution of 100 ul LF2000 in25 ul DMEM then both mixed together gently to make 250 ul total mixerwhich incubated 5-10 min at RT to form a Lipid-mRNA complex beforeinjection. The lipoplex from both Luciferase mRNA fully modified with1-methylpseudouridine or 1-methylpseudouridine and 5-methylcytosine wereadministered intranasally (I.N.) to Balb-C mice at a dose of 0.5 mg/kg.

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measured.The background signal was about 4.8+05 p/s. The background flux wasabout 6.3+05 p/s. The results of the imaging of Luc-G5-Lipoplex orLuc-G2-Lipoplex vs. Vehicle are shown in the Table 183.

TABLE 183 Luciferase expression after intranasal dosing TimeLuc-G5-Lipoplex Luc-G2-Lipoplex Vehicle (PBS) Route Point Flux (p/s)Flux (p/s) Flux (p/s) I.N.  2 hrs 8.37E+05 9.58E+05 3.87E+05 I.N.  8 hrs8.42E+05 7.11E+05 5.57E+05 I.N. 24 hrs 5.74E+05 5.53E+05 4.89E+05

Example 119 Intranasal Lung Delivery of 1-methylpseudouridine or5-methylcytosine and 1-methylpseudouridine Modified Luciferase mRNAformulated in PBS

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 33907; polyAtail of approximately 140 nucleotides not shown in sequence; 5′cap,Cap1) fully modified with 1-methylpseudouridine (Luc-G5-Buffer(PBS)) orfully modified with 1-methylpseudouridine and 5-methylcytosine(Luc-G2-Buffer(PBS)) formulated in PBS (pH 7.4) were administeredintranasally (I.N.) to Balb-C mice at a dose of 7.5 mg/kg.

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measured.The background flux was about 4.8+05 p/s. The background flux was about6.3+05 p/s. The results of the imaging of Luc-G5-in Buffer vs. Vehicleare shown in the Table 184.

TABLE 184 Luciferase expression after intranasal dosing Luc-G5-in BufferLuc-G2-in Buffer Vehicle Time (PBS) (PBS) (PBS) Route Point Flux [p/s]Flux [p/s] Flux (p/s) I.N.  2 hrs 4.50E+05 9.58E+05 3.87E+05 I.N.  8 hrs7.12E+05 7.11E+05 5.57E+05 I.N. 24 hrs 4.47E+05 5.53E+05 4.89E+05

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

LENGTHY TABLE

The patent application contains a lengthy table section. A copy of thetable is available in electronic form from the USPTO web site. Anelectronic copy of the table will also be available from the USPTO uponrequest and payment of the fee set forth in 37 CFR 1.19(b)(3).

We claim:
 1. An mRNA encoding a polypeptide of interest selected fromthe group consisting of SEQ ID NOs 4704-9203, wherein said mRNAcomprises a coding region selected from the group consisting of SEQ IDNOs: 9204-33882.
 2. The mRNA of claim 1, wherein the mRNA comprises atleast one untranslated region 5′ relative to the coding region and atleast one untranslated region 3′ relative to the coding region.
 3. ThemRNA of claim 2, wherein the 5′ untranslated region is heterologous tothe coding region of the mRNA.
 4. The mRNA of claim 2, wherein the 3′untranslated region is heterologous to the coding region of the mRNA. 5.The mRNA of claim 2, wherein the 5′ untranslated region and the 3′untranslated region are heterologous to the coding region of the mRNA.6. The mRNA of claim 2, wherein the mRNA comprises at least two stopcodons.
 7. A pharmaceutical composition comprising the mRNA of claim 1and a pharmaceutically acceptable excipient.
 8. The pharmaceuticalcomposition of claim 7, wherein the pharmaceutically acceptableexcipient is selected from a solvent, aqueous solvent, non-aqueoussolvent, dispersion media, diluent, dispersion, suspension aid, surfaceactive agent, isotonic agent, thickening or emulsifying agent,preservative, lipid, lipidoids liposome, lipid nanoparticle, core-shellnanoparticles, polymer, lipoplex, peptide, protein, cell, hyaluronidase,and mixtures thereof.
 9. The pharmaceutical composition of claim 8,where the pharmaceutical composition comprises a lipid and wherein saidlipid is selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5,C12-200, DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA and PEGylatedlipids and mixtures thereof.
 10. A method of producing a polypeptide ofinterest in a mammalian cell, tissue or organism comprising contactingsaid cell, tissue or organism with the mRNA of claim
 1. 11. The methodof claim 10, wherein the mRNA is formulated.
 12. The method of claim 11,wherein the formulation comprises a lipid which is selected from thegroup consisting of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5,C12-200, DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA, PEGylatedlipids and mixtures or combinations thereof.
 13. The method of claim 10,wherein the contacting is a route of administration selected from thegroup consisting of intramuscular, intradermal, intravenous andsubcutaneous.
 14. The method of claim 13, wherein the route ofadministration is intramuscular.
 15. The method of claim 10, wherein themRNA comprises at least one untranslated region 5′ relative to thecoding region and at least one untranslated region 3′ relative to thecoding region.
 16. The method of claim 15, wherein the 5′ untranslatedregion is heterologous to the coding region of the mRNA.
 17. The methodof claim 15, wherein the 3′ untranslated region is heterologous to thecoding region of the mRNA.
 18. The method of claim 15, wherein the 5′untranslated region and the 3′ untranslated region are heterologous tothe coding region of the mRNA.
 19. The method of claim 15, wherein themRNA comprises at least two stop codons.